Roles for dual endothelin-1/angiotensin ii receptor (dear) in hypertension and angiogenesis

ABSTRACT

The present application is directed to the identification of mutations and/or polymorphisms in the Dual Endothelin-1/Angiotensin II Receptor (Dear) that indicate susceptibility to, or show current affliction with, hypertension. Additionally, the present invention discloses methods for the modulation of angiogenesis via the regulation of Dear.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. Utility applicationSer. No. 11/667,713, which is a 371 National Phase Entry Application ofInternational Application PCT/US2005/041594, filed Nov. 15, 2005, whichdesignated the U.S. and which claims benefit under 35 USC 119(e) of theU.S. provisional application No. 60/628,447 filed on Nov. 16, 2004 andU.S. provisional application No. 60/694,268 filed on Jun. 27, 2005, thecontent of which is incorporated herein by reference in its entirety.

GOVERNMENT SUPPORT

This invention was made with Government Support under Contract No.HL69937 awarded by the National Institutes of Health. The Government hascertain rights in the invention.

FIELD OF THE INVENTION

The present application is directed to the identification of mutationsand/or polymorphisms in the Dual Endothelin-1/Angiotensin II Receptor(Dear) that indicate susceptibility to, or show current affliction with,hypertension. Additionally, the present invention discloses methods forthe modulation of angiogenesis via the regulation of Dear.

BACKGROUND OF THE INVENTION

The Dual Endothelin-1/Angiotensin II Receptor (Dear) was originallyisolated from an adult rat brain cDNA library using an AngII antisenseoligonucleotide probe and also, independently, with an ET-1oligonucleotide, see Molecular Medicine 4: 96-108, 1998. Structuralanalysis of the receptor revealed putative single predictedtransmembrane domain and distinct ET-1 and AngII putative bindingdomains. Functional analysis has shown that both ET-1 and AngII bind toDear and induce coupling to a Ca2+ mobilizing transduction system.

ET-1 is a potent vasoconstrictor peptide involved in diversephysiological functions such as blood pressure regulation, mitogenesisand apoptosis (Lariviere, R. et al. Can J Physiol Pharmacol. 81, 607-621(2003), and angiogenesis (Salani, D. et al. Am. J. Pathol. 157,1537-1547 (2000); Sullivan, D. C. & Bicknell, R. British Journal ofCancer 89, 228-231 (2003)), and has been implicated in severalpathophysiological conditions such as hypertension, cardiac failure(Lariviere, R. et al. Can J Physiol Pharmacol. 81, 607-621 (2003);Ikeda, T. et al. Hypertension 34, 514-519 (1999); Touyz, R. M. &Schiffrin, E. L. Can J Physiol Pharmacol. 81, 533-541 (2003)), and morerecently tumor angiogenesis, invasion and metastases (Bagnato, A. &Spinella, F. Trends in Endocrinology and Metabolism 14, 44-50 (2002);Grant, K., Loizidou, M. & Taylor, I. British Journal of Cancer 88,163-166 (2003)).

AngII exhibits similar physiological responses to ET-1, such as bloodpressure regulation, proliferation, apoptosis and angiogenesis(Watanabe, T. et al. Hypertension 45, 163-169 (2005), and has also beenimplicated in tumor angiogenesis (Escobar, E. et al. Curr Vasc Pharmacol2, 385-399 (2004)). Separate receptors have been identified for bindingby either ET-1 or AngII which are believed to be responsible for thephysiological responses observed.

Accordingly, despite known roles for ET-1 and AngII, the role of Dear iscurrently unknown. It is believed that Dear regulates pathways distinctfrom those triggered by either ET-1 or AngII binding to ET_(A), ET_(B)or AT1 and AT2 receptors respectively. However, due to its ability tobind to both ET-1 and AngII, and the important role these molecules playin angiogenesis, hypertension and tumor progression, a betterunderstanding of Dear's role is needed. The present invention disclosesnewly discovered roles for Dear and presents methods to screen for,diagnose, prognose and treat various diseases and disorders such ashypertension, pathological angiogenesis and tumor growth/metastasis.

The genomes of all organisms undergo spontaneous mutation in the courseof their continuing evolution, generating variant forms of progenitorgenetic sequences (Gusella, Ann. Rev. Biochem. 55, 831-854 (1986)). Avariant form may confer an evolutionary advantage or disadvantagerelative to a progenitor form or may be neutral. In some instances, avariant form confers an evolutionary advantage to the species and iseventually incorporated into the DNA of many or most members of thespecies and effectively becomes the progenitor form. However, oftentimes the variant form confers a disadvantage that may make anindividual susceptible to certain diseases or disorders. Anunderstanding of these variants may provide for better diagnosis ofexisting diseases or disorders, prognosis of the risk of obtainingcertain diseases or disorders, and improved, more targeted treatments.

The knowledge of specific mutations and/or polymorphisms that aredisease or disorder associated help identify patients most suited totherapy with particular pharmaceutical agents (this is often termed“pharmacogenetics”). Pharmacogenetics can also be used in pharmaceuticalresearch to assist the drug selection process. Polymorphisms are used inmapping the human genome and to elucidate the genetic component ofdiseases. The following references show background details onpharmacogenetics and other uses of polymorphism detection: Linder et al.(1997), Clinical Chemistry, 43, 254; Marshall (1997), NatureBiotechnology, 15, 1249; International Patent Application WO 97/40462,Spectra Biomedical; and Schafer et al. (1998), Nature Biotechnology, 16,33.

I. Hypertension

Hypertension, or high blood pressure, is the most common chronic illnessin America. The American Heart Association estimates that more than 62million Americans over the age of six suffer from high blood pressure,and that only a minority of these people have their blood pressure undercontrol. Left untreated, hypertension can lead to stroke, heart attack,kidney damage, congestive heart failure, and death. Uncontrolledmild-to-moderate hypertension will reduce the life expectancy of atypical 35-year-old person by 16 years. Even the mildest form of highblood pressure, “borderline hypertension,” can cut one's life span by afew years and impact negatively on the quality of life.

The existence of a genetic component to hypertension is known from twinstudies, which have revealed a greater concordance of blood pressure inmonozygotic twins than in dizygotic twins. Similarly, biologicalsiblings show greater concordance of blood pressure than adoptivesiblings raised in the same household. Such studies have suggested thatup to about 40% of the variations in blood pressure in the populationare genetically determined. However, to date, a reliable genetic markerfor hypertension has not been identified. Although significant gainshave been made with respect to treatment, hypertension prevails as amajor risk factor for heart and kidney disease, and stroke prompting thelowering of the BP level at which to start treatment.

Thus, a genetic marker for predicting one's susceptibility tohypertension is needed, as well as, a reliable method to diagnosehypertension is needed. Additionally, treatment strategies targeting thenormalization of mutant genes contributing to genetic hypertension(hypertension genes) are needed.

II. Angiogenesis

Angiogenesis is a process of tissue vascularization that involves boththe growth of new developing blood vessels into a tissue(neo-vascularization) and co-opting of existing blood vessels to atarget site. Blood vessels are the means by which oxygen and nutrientsare supplied to living tissues and waste products are removed fromliving tissue. Angiogenesis can be a critical biological process. Forexample, angiogenesis is essential in reproduction, development andwound repair. Conversely, inappropriate angiogenesis can have severenegative consequences. For example, it is only after solid tumors arevascularized as a result of angiogenesis that the tumors have asufficient supply of oxygen and nutrients that permit it to grow rapidlyand metastasize.

Angiogenesis-dependent diseases and disorders are those diseases anddisorders affected by vascular growth. Such diseases represent asignificant portion of diseases for which medical treatment is sought,and include inflammatory disorders such as immune and non-immuneinflammation, chronic articular rheumatism and psoriasis, disordersassociated with inappropriate or inopportune invasion of vessels such asdiabetic retinopathy, macular degeneration, neovascular glaucoma,restenosis, capillary proliferation in atherosclerotic plaques andosteoporosis, and cancer associated disorders, such as solid tumors,solid tumor metastases, angiofibromas, retrolental fibroplasia,hemangiomas, Kaposi sarcoma, cancers which require neovascularization tosupport tumor growth, etc.

While methods to inhibit unwanted angiogenesis are known, few haveproven clinically useful. For example, a number of therapeuticstrategies exist for inhibiting aberrant angiogenesis, which attempt toreduce the production or effect of VEGF. For example, anti-VEGF or VEGFreceptor antibodies (Kim E S et al. (2002), PNAS USA 99: 11399-11404),and soluble VEGF “traps” which compete with endothelial cell receptorsfor VEGF binding (Holash J et al. (2002), PNAS USA 99: 11393-11398) havebeen developed. Classical VEGF “antisense” or aptamer therapies directedagainst VEGF gene expression have also been proposed (U.S. publishedapplication 2001/0021772 of Uhlmann et al.). The anti-angiogenic agentsused in these and similar non-VEGF targeted therapies have typicallybeen unsuccessful. The results achieved with available anti-angiogenictherapies have therefore been generally unsatisfactory.

Thus, methods to reduce or eliminate unwanted angiogenesis are needed.

Conversely, in situations where angiogenesis is desired, such as, forexample, reproduction, development, wound repair and areas of ischemiaor infarction, the stimulation of angiogenesis is useful. Currentmethods to initiate or up-regulate angiogenesis have also typically beenclinically unsuccessful and are thus needed.

Furthermore, because ET-1 is also associated with breast cancer growthand pro-malignant potential, inhibition of Dear will also be useful indecreasing tumor growth and potential to metastasize, independent of itseffects on angiogenesis.

SUMMARY OF THE INVENTION

The inventors of the present invention have discovered that mutationsand/or polymorphisms in Dear that enhances the expression and/or theaffinity of ET-1 binding to Dear can accurately predict susceptibilityto hypertension. Mutations and/or polymorphisms in Dear and humanhomologues thereof are encompassed in the present invention. Thus, inone embodiment of the present invention, it is possible to predictsusceptibility to hypertension, to diagnose current hypertension and/orprovide prognosis of the hypertension by analyzing Dear gene and/orprotein. The presence of a mutation and/or polymorphism that (1)enhances Dear expression and/or (2) enhances the affinity of ET-1binding to Dear, compared to a wild type control, is indicative of one'ssusceptibility to and/or current affliction with hypertension.

In addition, Dear plays a role in angiogenesis, tumor growth andpro-malignant potential. In particular, the inventors have shown thatinhibitors of Dear, such as anti-Dear antibodies, decreased tumorprogression and pro-malignant potential in both a rat and mouse model ofcancer. Thus, in another embodiment of the present invention, methods toinhibit angiogenesis and/or tumor growth and/or pro-malignant potentialare disclosed. In this embodiment, an individual is administered acompound that inhibits Dear activation, such as for example, a smallmolecule inhibitor, competitive inhibitor, antibody, antibody fragment,sirna, aptamer, etc. Preferably, these are used in conjunction withinhibitors to other angiogenesis-associated agents such as VEGF,placental growth factors etc.

Non-limiting examples of pathological angiogenesis or disorders treatedby the methods of the present invention include, inflammatory disorderssuch as immune and non-immune inflammation, chronic articular rheumatismand psoriasis, disorders associated with inappropriate or inopportuneinvasion of vessels such as diabetic retinopathy, macular degeneration,neovascular glaucoma, restenosis, capillary proliferation inatherosclerotic plaques and osteoporosis, and cancer associateddisorders, such as solid tumors, solid tumor metastases, angiofibromas,retrolental fibroplasia, hemangiomas, Kaposi sarcoma and the likecancers which require neovascularization to support tumor growth. In apreferred embodiment of the present invention, the methods are directedto inhibiting tumor angiogenesis and/or pro-malignant potential in amammal with cancer, such as, for example, breast cancer.

In a related embodiment, the present invention discloses methods tostimulate angiogenesis in tissues in need thereof. In this embodiment,activators of Dear are administered to an individual, such as, forexample, small molecules, antibodies, antibody fragments, or otheractivators known to those of skill in the art.

As an example, stimulation of angiogenesis may be beneficial indiabetes-induced ischemia, poor circulation, myocardial infarction,aortic aneurysm, arterial disease of the lower extremities,cerebrovascular disease, etc.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C. Molecular characterization of Dahl S and Dahl R Dearvariants. (FIG. 1A) Comparative nucleotide sequence of Dahl S and Dahl RcDNAs spanning the T²⁸¹⁴ (Dahl S)/C²⁸¹⁴ (Dahl R) and T²⁹⁰¹ (DahlS)/C²⁹⁰¹ (Dahl R) nucleotide transitions. Amino acid substitutionsresulting from the corresponding nucleotide transitions detected S44substitution in Dahl R Dear for P44 and M74 substitution in Dahl R Dearfor T74 (amino acid numbering as per Ruiz-Opazo et al. 1998) FIG. 1Adiscloses SEQ ID NOS: 17-22, respectively, in order of appearance. FIG.1B shows schematic structure of the Dahl R Dear (SEQ ID NO: 23. Thefollowing functional domains are highlighted: putative AngII bindingsite, AngII (aa 41-48); ET-1 binding site, ET-1 (aa 60-67); amino acidS44 and M74 substituted in the Dahl S Dear by P44 and T74 respectively;potential cAMP-dependent protein kinase phosphorylation sites (S91, T108in green); a potential internalization recognition sequence (IRS) (FIG.1C) Western blot analysis detects equivalent levels of Dahl S(S) andDahl R(R) Dear variants in Dahl S and Dahl R rat kidney membranesisolated from male and female rats. MW, 14.4 kDa molecular weightmarker.

FIGS. 2A-2C. Functional characterization of Dahl S and Dahl R Dearvariants. Saturation binding curves of ligand binding studies of Dahl S(∘) and Dahl R (•) Dear expressed in Cos1 cells with radiolabeled¹²⁵I-AngII (FIG. 2A) and ¹²⁵I-ET-1 (FIG. 2B). Values are presented asMean±standard deviation from five independent experiments. (FIG. 2C)Detection of the 14 kDa Dear protein (

) by western blot analysis (ab) of Dahl R (Kid-R) and Dahl S (Kid-S)kidney (Kid) membranes; control non-transfected Cost cell membranes(Cos1-c), Cost cell membranes expressing the Dahl R S44P/M74T variant(Cos1-R) and Cost cell membranes expressing the Dahl S S44/M74 variant(Cos1-S). ¹²⁵I-AngII west-western blot analysis (*AngII) detects bindingonly to Dahl S kidney membranes (Kid-S) and Cos1 cell membranesexpressing the Dahl S S44/M74 variant (Cos1-S) while ¹²⁵I-ET-1west-western blot analysis (*ET-1) reveals binding to both Dahl R(Kid-R) and Dahl S (Kid-S) kidney membranes as well as to Cos1 cellmembranes expressing the Dahl R S44P/M74T (Cos1-R) and Dahl S S44/M74(Cos1-S) molecular variants.

FIGS. 3A-3D. Scatchard plots of saturation data for Dahl S and Dahl RDear variants. Scatchard plots of ¹²⁵I-AngII (FIG. 3A) and ¹²⁵I-ET-1(FIG. 3B, FIG. 3C) saturation binding data of Dahl S (∘) and Dahl R (•)Dear expressed in Cos1 cells. FIG. 3D: Saturation binding curves ofligand binding studies of mouse Dear expressed in Cos-1 cells. ◯,mean±sem ¹²⁵I-ET-1 binding; , mean±sem ¹²⁵I-AngII binding.

FIGS. 4A-4C. Detection and genetic analysis of Dear variants. (FIG. 4A)Detection of the Dear gene variants by single strand conformationpolymorphism (SSCP) analysis in different rat strains. A 137 bp PCRproduct spanning the S44P substitution reveals the S44P/M74T variant inDahl R(R) and LEW strains while the S44/M74 variant is detected in DahlS(S), BN, WKY and SHR genomic DNAs. F1 denotes F1 [RxS] subjects.Interval mapping with bootstrap-analysis for chromosome-2 in male (FIG.4B) and female (FIG. 4C) cohorts using Map Manager QTXb17 program.Horizontal lines (—) mark LRS values for significance of linkage. For(FIG. 4B) from top to bottom: LRS=18.4 (LOD=4.00) for highlysignificant, LRS=10.6 (LOD=2.30) for significant, LRS=4.1 (LOD=0.89) forsuggestive; for (FIG. 4C) from top to bottom: LRS=16.6 (LOD=3.61) forhighly significant, LRS=9.9 (LOD=2.15) for significant, LRS=3.9(LOD=0.85) for suggestive. — Likelihood ratio statistic; — regressioncoefficient for additive effect; — regression coefficient for dominanceeffect. Histograms represent the bootstrap-based confidence intervalsfor the detected QTLs.

FIG. 5 shows Dear expression and schematic representation of thetargeting vector. FIG. 5A shows a restriction map of wild type 129SVJmouse Dear (WT allele), the Dear targeting vector for homologousrecombination (KO construct), and the mutant allele. (1) is a probe forSouthern-blot analysis, which is a 1.5 kb S-B restriction fragment whichdetects a 5.2 kb SphI restriction digest fragment in targeted allele (2)and a 8.0 kb fragment in the wild type allele (3); P1 (4) is a forwardprimer flanking integration site; P2 (5) is a reverse pGKNeo-specificprimer; successful targeting event yields expected 5.5 kb P1-P2 PCRproduct. S, SacI; B, BamHI; N, NsiI restriction enzymes.

FIG. 5B shows a Northern blot analysis which detects mouse Dear mRNA inall tissues tested with higher levels in kidney and aorta. The threedifferent-sized Dear mRNAs most likely represent differentpolyadenylation signals. 28S and 18S ribosomal markers are noted to theleft. H, heart, B, brain, K, kidney, Li, liver, Sp, spleen, Lu, lung,Ao, aorta, Te, testis, Ut, uterus.

FIG. 5C shows a Southern blot analysis for detection ofDear-inactivation in mice by homologous recombination. Evidence ofinactivation is characterized by predicted 5.2 kb SphI restrictiondigest fragment of genomic DNA detected as a lower hybridizing band inheterozygous (+/−) DNA samples compared with absence in wild type (+/+)samples.

FIG. 5D shows PCR genotyping of E11.5 mouse embryos. Results (lowerpanel) show a 153 bp allele in wild type, Dear^(+/+) (+/+) andheterozygous Dear^(+/−) (+/−), but not in Dear^(−/−) (−/−) embryos. Theupper panel shows inactivated (5.5 kb) allele in Dear^(+/−) andDear^(−/−) but not in Dear^(+/+) embryos.

FIG. 5E shows deduced amino acid sequence for mouse Dear cDNA (bottomsequence; SEQ ID NO: 24) compared with rat Dear sequence (SEQ ID NO: 2);Ab, peptide sequence used for anti-Dear antibody; AngII, angiotensin IIbinding site¹, ET-1, endothelin-1 binding site¹, TM-1, predictedtransmembrane domain; IRS, internalization recognition sequence; (−),identical sequence; (*), adjusted gap for better alignment; boldlettering denotes non-conservative amino acid differences.

FIG. 5F shows a Western blot analysis of Dear^(+/+) (+/+) and Dear^(+/−)(+/−) deficient mouse kidney membranes using the anti-mouse Dearspecific anti-peptide antibody (upper panel); presence of a non-specificcross-reacting high molecular weight protein demonstrates equal amountsof protein analyzed (lower panel).

FIGS. 5G-5I show analysis of blood pressure (FIG. 5G), heart rate (FIG.5H) and body weight (FIG. 5I) in heterozygous Dear^(+/−) adult mice.Systolic blood pressure (SBP, mmHg) and mean heart rate (bpm, in beatsper minute) in Dear^(+/+) (□) and Dear^(+/−) (▪) deficient male (M) andfemale (F) mice. Body weight in grams (g) comparing male Dear^(+/+) (⋄)and Dear^(+/−) (♦) mice, and female Dear^(+/+) (◯) and Dear^(−/−) ()mice from 4-6 months (m) of age. *, P<0.05; **, P<0.01.

FIGS. 6A-I show comparative anatomical analysis of Dear^(+/+) (leftside) and Dear^(−/−) (right side) mouse embryos. FIG. 6A shows adjacentE12.5 embryos revealing prominent yolk-sac collecting vessels inDear^(+/+) (left side) but absent in the smaller Dear^(−/−) embryo(right side); both embryos still attached to placentas respectively.

FIG. 6B shows adjacent E11.5 mouse embryos revealing well-developedyolk-sac collecting vessels in Dear^(+/+) while absent in Dear^(−/−)embryo.

FIG. 6C shows that compared to E10.5 Dear^(+/+), E10.5 Dear^(−/−) mouseembryo exhibits a lack of yolk-sac collecting vessels; embryotranslucency allows detection of incomplete vascular network, bloodfilled heart and some cranial region vascularization.

FIG. 6D shows adjacent E12.5 mouse embryos distinguishing normalDear^(+/+) from darkened, resorbed Dear^(−/−) embryo.

FIG. 6E shows E11.5 Dear^(−/−) mouse embryo exhibiting a hypoplasticphenotype compared with age-matched, larger dysmorphic null phenotype inFIG. 6B.

FIG. 6F shows E10.5 Dear^(−/−) mouse embryo exhibiting a hypoplasticphenotype compared with a slightly larger E10.5 dysmorphic nullphenotype in FIG. 6C.

FIG. 6G shows high magnification of E9.5 Dear^(+/+) (left panel) mouseembryo showing vascular network development from cranial to caudalregion with prominent blood-filled dorsal aortae (∇) and heart (→), incontrast to E10.5 Dear^(−/−) embryo (right panel) with rudimentary andabnormal vascular plexus in cranial region, an isolated blood filledheart (4), and non-detection blood-filled dorsal aortae.

FIG. 6H shows an analysis of mouse embryos revealing distinctblood-filled cardiac ventricles and vascular network throughout the bodyin the larger Dear^(+/+) embryo, in contrast to Dear^(−/−) embryo withan enlarged single-chamber blood-filled heart, minimal peripheralvascular network, absent eye pigmentation, and abnormal brain regiondevelopment.

FIG. 6I shows cleared, fixed E11.5 mouse embryos shown in FIG. 6B,revealing marked developmental delay in Dear^(−/−) embryo particularlyin brain region development and heart chamber formation.

FIGS. 7A-7L shows histologic analysis of Masson-trichrome stained mouseembryos. FIG. 7A: E10.5 Dear^(−/−) yolk sac revealing sparse bloodislands in primary vascular plexus with a dilated vessel (→), but absentcollecting vessels. FIG. 7B: E10.5 Dear^(+/+) embryo yolk-sac revealinglarge blood island-filled collecting vessels (→) and primary vascularplexus. FIG. 7C: Analysis of E12.5 Dear^(−/−) embryos reveals abnormalhyper-convoluted neuroepithelium with disorderly demarcation of majorbrain regions. Central ventral section is devoid of organogenesis withno recognizable liver and gut differentiation. FIG. 7D: Analysis oflittermate E12.5 Dear^(+/+) embryo contrasts the dysmorphic phenotype inthe Dear^(−/−) mutant. Note prominent organogenesis: gut, liver, heart,brain and dorsal aorta. FIG. 7E: High magnification of Dear−/−neuroepithelial segment (proximal * in FIG. 7C) revealing thin-walledperineural vessels (→) and a hyper-cellular neuroepithelium. FIG. 7F: Incontrast, high magnification of Dear+/+ neuroepithelial segment(proximal * in FIG. 7C) revealing perineural vessels (→) filled withblood islands and exhibiting relatively thicker walls. FIG. 7G: Highermagnification of Dear−/− neuroepithelium segment (distal * in FIG. 7C)showing marked cellularity with poor differential layering, absentpenetrating capillaries, although a few nucleated rbcs are detectedwithin the neuroepithelium. FIG. 7H: Higher magnification of Dear^(+/+)neuroepithelium (distal * in FIG. 7D) revealing differential layeringand numerous penetrating capillaries (→) with nucleated rbcs. FIGS.7I-J: Analysis of fetal-placental connections (f-pl con/xn) (bar=160 μm)and FIGS. 7K-L: fetal-placental junctions (f-pl jxn) in E11.5 embryosshows abnormal vascular development and decreased embryonic blood cellsin Dear^(−/−), (bar=20 μm).

FIGS. 8A-F show histological analysis of Dear^(+/+) (+/+) (FIGS. 8A, 8Cand 8E) and Dear^(−/−) (−/−) mouse embryos (FIGS. 8B, 8D and 8F).Masson-trichrome stained E11.5 embryos showing deficient development ofdorsal aorta (da), vasculature (vasc) and yolk sac, as well as heart andbrain in Dear^(−/−) (bar=160 μm).

FIGS. 9A-F show smooth muscle cell a-actin immunostaining of E12.5 mouseembryos (embryo (FIG. 9A-B); bar=160 μm) demarcating angiogenesis(angiog; FIG. 9C-D) in perineural region and formation of vascularnetwork (vasc net; FIG. 9E-F) in the caudal region detects deficientvascular development in Dear^(−/−) embryos; bar=20 μm.

FIGS. 10A and 10B show analysis of mouse dear expression pattern in wildtype (+/+) E9.5 and E12.5 embryos detects Dear expression in the heart,extra-embryonic and embryonic vasculature, and neural tube. sc, spinalcord, ao, aorta, bi, blood islands; neuroepith, neuroepithelium; bar=20μm.

FIGS. 11A-11E show analysis of Dear-inhibition on tumor growth. InDear^(+/−) mice (▪), decreased tumor mass (mg) (FIG. 11A) and tumorvolume (mm³) (FIG. 11B) of melanoma cell-induced subcutaneous tumorswere observed in females but not in males compared with age-matchedDear^(+/+) control mice (□). FIG. 11C: Anti-rat Dear anti-peptidespecific antibody treatment (◯) results in decreased tumor volume inradiation-induced rat mammary tumors. FIG. 11D: Anti-rat Dear DNAvaccine treatment (⋄) also results in decreased tumor volume inradiation-induced rat mammary tumors. FIG. 11E: Representativehistological analysis of Masson-trichrome stained tumor sectionscomparing mock-treated (mock-Rx) vector controls, anti-Dear anti-peptidespecific antibody treatment (ab-Rx) and anti-rat Dear DNA-vaccine(DNA-vac) shows changes in tumor pattern, microvascular invasion andnuclear grade in anti-Dear treated tumors; bar=20 μm. Values arepresented as mean±sem. *, P<0.05; **, P<0.01;***, P, <0.001

DETAILED DESCRIPTION OF THE INVENTION I. Hypertension

In one embodiment of the present invention, a method for determining anindividual's susceptibility to hypertension is disclosed, as well as adiagnostic and/or prognostic method to determine the condition of thehypertension. An individual is screened for mutations and/orpolymorphisms in Dear that correlate to an increase expression of Dearand/or enhancement of Dear-activation or Dear-signaling by AngII, ET-1,VEGF-signal peptide (VEGFsp) or other Dear-ligand as compared to acontrol. The presence of such a mutation and/or polymorphism in the testsample, as compared to the normal control, is indicative of anindividual's susceptibility to hypertension. The presence of such amutation and/or polymorphism in the test sample, as compared to thenormal control, can also be indicative of a current state ofhypertension.

As used herein, the term Dear encompasses Dear and human homologuesthereof. In one embodiment, the term “human homologue to Dear” refers toa DNA sequence that has at least about 50% homology to SEQ ID NO:1 andmore preferably at least about 60% homology or identity, including allintervals up (i.e. 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, etc.).In one embodiment, the term “human homologue to Dear protein” refers toan amino acid sequence that has 50% homology to SEQ ID NO: 2, morepreferably at least about 60% homology, still more preferably, at leastabout 70% homology, even more preferably, at least about 75% homology,yet more preferably, at least about 80% homology, even more preferablyat least about 85% homology, still more preferably, at least about 90%homology, and more preferably, at least about 95% homology, intervals ofthe same are also encompassed (i.e., 55%, 60%, 70%, 75%, 80%, 85%, 90%,95%, 98%, etc.).

“Homology” or “identity” or “similarity” refers to sequence similaritybetween two peptides or between two nucleic acid molecules. Homology andidentity can each be determined by comparing a position in each sequencewhich may be aligned for purposes of comparison. When an equivalentposition in the compared sequences is occupied by the same base or aminoacid, then the molecules are identical at that position; when theequivalent site occupied by the same or a similar amino acid residue(e.g., similar in steric and/or electronic nature), then the moleculescan be referred to as homologous (similar) at that position. Expressionas a percentage of homology/similarity or identity refers to a functionof the number of identical or similar amino acids at positions shared bythe compared sequences. A sequence which is “unrelated” or“non-homologous” shares less than 40% identity, though preferably lessthan 25% identity with a sequence of the present application.

In comparing two sequences, the absence of residues (amino acids ornucleic acids) or presence of extra residues also decreases the identityand homology/similarity. The term “homology” describes a mathematicallybased comparison of sequence similarities which is used to identifygenes or proteins with similar functions or motifs. The nucleic acid andprotein sequences of the present application may be used as a “querysequence” to perform a search against public databases to, for example,identify other family members, related sequences or homologs. Suchsearches can be performed using the NBLAST and XBLAST programs (version2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLASTnucleotide searches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to nucleic acidmolecules of the application. BLAST protein searches can be performedwith the XBLAST program, score=50, wordlength=3 to obtain amino acidsequences homologous to protein molecules of the application. To obtaingapped alignments for comparison purposes, Gapped BLAST can be utilizedas described in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., XBLAST and BLAST)can be used. See ncbi web site at nih.gov.

As used herein, “identity” means the percentage of identical nucleotideor amino acid residues at corresponding positions in two or moresequences when the sequences are aligned to maximize sequence matching,i.e., taking into account gaps and insertions. Identity can be readilycalculated by known methods, including but not limited to thosedescribed in (Computational Molecular Biology, Lesk, A. M., ea., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and—14Genome Projects, Smith, D. W., ea., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math.,48: 1073 (1988)). Methods to determine identity are designed to give thelargest match between the sequences tested. Moreover, methods todetermine identity are codified in publicly available computer programs.Computer program methods to determine identity between two sequencesinclude, but are not limited to, the GCG program package (Devereux, J.,et al., Nucleic Acids Research 112(1): 387 (1984)), BLASTP, BLASTN, andFASTA (Altschul, S. F. et al., J. I Molec. Biol. 215: 403-410 (1990) andAltschul et al. Nuc. Acids Res. 25: 3389-3402 (1997)). The BLAST Xprogram is publicly available from NCBI and other sources (BLAST Manual,Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., etal., J. Mol. Biol. 215: 403-410 (1990)). The well known Smith Watermanalgorithm may also be used to determine identity.

Once an individual is known to be susceptible to hypertension, currentlyavailable methods to reduce or prevent a rise in blood pressure may beused. Examples of currently available methods to reduce and/or preventhypertension include, for example, diet, exercise, multidrug regimensincluding combinations of antihypertensive drugs such as beta blockers,diuretics, calcium antagonists, angiotensin II active agents, heartrate-reducing nondihydropyridine calcium antagonists, andangiotensin-converting enzyme (ACE) inhibitors. Alternatively, compoundsthat modulate Dear function may be administered. Where the individualalready has hypertension knowing the basis for that hypertension can beused to determine treatment regime.

Methods to detect the presence or absence of mutations and/orpolymorphisms in genes such as Dear are known to those of skill in theart and certain embodiments are as follows:

Preparation of Samples

Mutations and/or polymorphisms are detected in a target nucleic acidfrom an individual being analyzed. For example, for assay of genomicDNA, virtually any biological sample (other than pure red blood cells)is suitable. For example, convenient tissue samples include whole blood,semen, saliva, tears, urine, fecal material, sweat, buccal, skin andhair. For assay of cDNA or mRNA, the tissue sample must be obtained froman organ in which the target nucleic acid is expressed, see for example,FIG. 5B, which shows Dear expression in heart, brain, kidney, liver,spleen, lung, aorta, testis, and uterus.

Many of the methods described below require amplification of DNA fromtarget samples. This can be accomplished by e.g., PCR. See generally PCRTechnology: Principles and Applications for DNA Amplification (ed. H. A.Erlich, Freeman Press, NY, N.Y., 1992); PCR Protocols: A Guide toMethods and Applications (eds. Innis, et al., Academic Press, San Diego,Calif., 1990); Mattila et al., Nucleic Acids Res. 19, 4967 (1991);Eckert et al., PCR Methods and Applications 1, 17 (1991); PCR (eds.McPherson et al., IRL Press, Oxford); and U.S. Pat. No. 4,683,202 (eachof which is incorporated by reference for all purposes).

Other suitable amplification methods include the ligase chain reaction(LCR) (see Wu and Wallace, Genomics 4, 560 (1989), Landegren et al.,Science 241, 1077 (1988), transcription amplification (Kwoh et al.,Proc. Natl. Acad. Sci. USA 86, 1173 (1989)), and self-sustained sequencereplication (Guatelli et al., Proc. Nat. Acad. Sci. USA, 87, 1874(1990)) and nucleic acid based sequence amplification (NASBA). Thelatter two amplification methods involve isothermal reactions based onisothermal transcription, which produce both single stranded RNA (ssRNA)and double stranded DNA (dsDNA) as the amplification products in a ratioof about 30 or 100 to 1, respectively.

Detection of Mutations and/or Polymorphisms in Target DNA

The identity of bases occupying mutated or polymorphic sites can bedetermined in an individual (e.g., a patient being analyzed) by severalmethods, which are described in turn.

Allele-Specific Probes

The design and use of allele-specific probes for analyzing mutationsand/or polymorphisms is described by e.g., Saiki et al., Nature 324,163-166 (1986); Dattagupta, EP 235,726, Saiki, WO 89/11548.Allele-specific probes can be designed that hybridize to a segment oftarget DNA from one individual but do not hybridize to the correspondingsegment from another individual due to the presence of different mutatedor polymorphic forms in the respective segments from the twoindividuals. Hybridization conditions should be sufficiently stringentthat there is a significant difference in hybridization intensitybetween alleles, and preferably an essentially binary response, wherebya probe hybridizes to only one of the alleles. Some probes are designedto hybridize to a segment of target DNA such that the polymorphic sitealigns with a central position (e.g., in a 15 mer at the 7 position; ina 16 mer, at either the 8 or 9 position) of the probe. This design ofprobe achieves good discrimination in hybridization between differentallelic forms.

Allele-specific probes are often used in pairs, one member of a pairshowing a perfect match to a reference form of a target sequence and theother member showing a perfect match to a variant form. Several pairs ofprobes can then be immobilized on the same support for simultaneousanalysis of multiple mutations and/or polymorphisms within the sametarget sequence.

Tiling Arrays

The mutations and/or polymorphisms can also be identified byhybridization to nucleic acid arrays, some example of which aredescribed by WO 95/11995 (incorporated by reference in its entirety forall purposes). The same array or a different array can be used foranalysis of characterized mutations and/or polymorphisms. WO 95/11995also describes subarrays that are optimized for detection of a variantform of a precharacterized mutation and/or polymorphism. Such a subarraycontains probes designed to be complementary to a second referencesequence, which is an allelic variant of the first reference sequence.The second group of probes is designed by the same principles asdescribed above except that the probes exhibit complementarily to thesecond reference sequence. The inclusion of a second group (or furthergroups) can be particularly useful for analyzing short subsequences ofthe primary reference sequence in which multiple mutations are expectedto occur within a short distance commensurate with the length of theprobes (i.e., two or more mutations within 9 to 21 bases).

Allele-Specific Primers

An allele-specific primer hybridizes to a site on target DNA overlappinga mutation and/or polymorphism and only primes amplification of anallelic form to which the primer exhibits perfect complementarily. SeeGibbs, Nucleic Acid Res. 17, 2427-2448 (1989). This primer is used inconjunction with a second primer which hybridizes at a distal site.Amplification proceeds from the two primers leading to a detectableproduct signifying the particular allelic form is present. A control isusually performed with a second pair of primers, one of which shows asingle base mismatch at the polymorphic site and the other of whichexhibits perfect complementarily to a distal site. The single-basemismatch prevents amplification and no detectable product is formed. Themethod works best when the mismatch is included in the 3′-most positionof the oligonucleotide aligned with the mutation and/or polymorphismbecause this position is most destabilizing to elongation from theprimer. See, e.g., WO 93/22456.

Direct-Sequencing

The direct analysis of the sequence of mutation and/or polymorphisms ofthe present invention can be accomplished using either the dideoxy-chaintermination method or the Maxam-Gilbert method (see Sambrook et al.,Molecular Cloning, A Laboratory Manual (2nd Ed., CSHP, New York 1989);Zyskind et al., Recombinant DNA Laboratory Manual, (Acad. Press, 1988)).

Denaturing Gradient Gel Electrophoresis

Amplification products generated using the polymerase chain reaction canbe analyzed by the use of denaturing gradient gel electrophoresis.Different alleles can be identified based on the differentsequence-dependent melting properties and electrophoretic migration ofDNA in solution. Erlich, ed., PCR Technology, Principles andApplications for DNA Amplification, (W. H. Freeman and Co, New York,1992), Chapter 7.

Single-Strand Conformation Mutation and/or Polymorphism Analysis

Alleles of target sequences can be differentiated using single-strandconformation mutation and/or polymorphism analysis, which identifiesbase differences by alteration in electrophoretic migration of singlestranded PCR products, as described in Orita et al., Proc. Nat. Acad.Sci. 86, 2766-2770 (1989). Amplified PCR products can be generated asdescribed above, and heated or otherwise denatured, to form singlestranded amplification products. Single-stranded nucleic acids mayrefold or form secondary structures which are partially dependent on thebase sequence. The different electrophoretic mobilities ofsingle-stranded amplification products can be related to base-sequencedifference between alleles of target sequences.

Antibody Detection Methods

According to another aspect of the present invention an antibodyspecific for an allelic variant (mutation and/or polymorphism) of humanDear polypeptide is used to detect the presence or absence of Dearmutations and/or polymorphisms.

Antibodies can be prepared using any suitable method. For example,purified polypeptide may be utilized to prepare specific antibodies. Theterm “antibodies” is meant to include polyclonal antibodies, monoclonalantibodies, and the various types of antibody constructs such as forexample F(ab′)2, Fab and single chain Fv. Antibodies are defined to bespecifically binding if they bind the allelic variant of Dear with a Kaof greater than or equal to about 10⁷ M-1. Affinity of binding can bedetermined using conventional techniques, for example those described byScatchard et al., Ann. N.Y. Acad. Sci., 51:660 (1949).

Polyclonal antibodies can be readily generated from a variety ofsources, for example, horses, cows, goats, sheep, dogs, chickens,rabbits, mice or rats, using procedures that are well-known in the art.In general, antigen is administered to the host animal typically throughparenteral injection. The immunogenicity of antigen may be enhancedthrough the use of an adjuvant, for example, Freund's complete orincomplete adjuvant. Following booster immunizations, small samples ofserum are collected and tested for reactivity to antigen. Examples ofvarious assays useful for such determination include those described in:Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold SpringHarbor Laboratory Press, 1988; as well as procedures such ascountercurrent immuno-electrophoresis (CIEP), radioimmunoassay,radioimmunoprecipitation, enzyme-linked immuno-sorbent assays (ELISA),dot blot assays, and sandwich assays, see U.S. Pat. Nos. 4,376,110 and4,486,530.

Monoclonal antibodies may be readily prepared using well-knownprocedures, see for example, the procedures described in U.S. Pat. Nos.4,902,614, 4,543,439 and 4,411,993; Monoclonal Antibodies, Hybridomas: ANew Dimension in Biological Analyses, Plenum Press, Kennett, McKearn,and Bechtol (eds.), (1980).

Monoclonal antibodies to variant forms of Dear can be produced usingalternative techniques, such as those described by Alting-Mees et al.,“Monoclonal Antibody Expression Libraries: A Rapid Alternative toHybridomas”, Strategies in Molecular Biology 3: 1-9 (1990) which isincorporated herein by reference. Similarly, binding partners can beconstructed using recombinant DNA techniques to incorporate the variableregions of a gene that encodes a specific binding antibody. Such atechnique is described in Larrick et al., Biotechnology, 7: 394 (1989).

Once isolated and purified, the antibodies may be used to detect thepresence of variant Dear in a sample using established assay protocols,see for example “A Practical Guide to ELISA” by D. M. Kemeny, PergamonPress, Oxford, England. As is known to those of skill in the art, asuitable control sample (i.e. a sample with wild type or non-mutatedand/or polymorphic Dear) is used as a control.

Also encompassed are methods for the determination of expression levels.For example, the overexpression of Dear is an indication that anindividual is susceptible to or is currently afflicted withhypertension. Methods for analyzing expression levels are known to thoseof skill in the art. In one aspect of the invention, Dear levels presentin a test biological sample are measured by analyzing the level of DearmRNA in a test sample and comparing this level to the level of Dear in acontrol sample. In another embodiment, Dear levels present in a testbiological sample are measured by contacting the test sample, orpreparation thereof, with an endogenous control 18S rRNA. A preferredembodiment of the present invention is the use of laser capturemicrodissection and RT-PCR for the analysis of Dear mRNA from tissuesamples. Laser capture microdissection is known to those of skill in theart and described, for example, in Simon et al. (1998) Trends inGenetics 14:272 and Emmert-Buck et al. (1996) Science 274:998-1001.

In another aspect of the invention, Dear levels present in a testbiological sample are measured by contacting the test sample, orpreparation thereof, with an antibody-based binding moiety thatspecifically binds to Dear protein, or to a portion thereof. Theantibody-based binding moiety forms a complex with Dear that can bedetected, thereby allowing the levels of Dear to be measured.

Any means known to those skilled in art can be used to asses Dearlevels. For example, in some embodiments Dear expression levels areassayed by measuring levels of Dear via mass spectrometry, ELISA, MR,CT, PET targeted at Dear or immunohistochemistry.

In a further embodiment, the invention provides for kits that comprisemeans for measuring Dear in a biological sample.

DEFINITIONS

“Dear”, “DEAR”, or “Dear” as used herein and throughout is the DualEndothelin-1/Angiotensin II Receptor.

Polymorphism refers to the occurrence of two or more geneticallydetermined alternative sequences or alleles in a population. Apolymorphic marker or site is the locus at which divergence occurs.Preferred markers have at least two alleles, each occurring at frequencyof greater than 1%, and more preferably greater than 10% or 20% of aselected population. A polymorphic locus may be as small as one basepair. Polymorphic markers include restriction fragment lengthpolymorphisms, variable number of tandem repeats (VNTR's), hypervariableregions, minisatellites, dinucleotide repeats, trinucleotide repeats,tetranucleotide repeats, simple sequence repeats, and insertion elementssuch as Alu. The first identified allelic form is arbitrarily designatedas the reference form and other allelic forms are designated asalternative or variant alleles.

The allelic form occurring most frequently in a selected population issometimes referred to as the wild type form. Diploid organisms may behomozygous or heterozygous for allelic forms. A dialletic polymorphismhas two forms. A triallelic polymorphism has three forms.

A single nucleotide polymorphism occurs at a polymorphic site occupiedby a single nucleotide, which is the site of variation between allelicsequences. The site is usually preceded by and followed by highlyconserved sequences of the allele (e.g., sequences that vary in lessthan 1/100 or 1/1000 members of the populations).

A single nucleotide polymorphism usually arises due to substitution ofone nucleotide for another at the polymorphic site. A transition is thereplacement of one purine by another purine or one pyrimidine by anotherpyrimidine. A transversion is the replacement of a purine by apyrimidine or vice versa. Single nucleotide polymorphisms can also arisefrom a deletion of a nucleotide or an insertion of a nucleotide relativeto a reference allele.

II. Angiogenesis A. Inhibiting Angiogenesis

In another embodiment of the present invention, a method of inhibitingangiogenesis in a tissue of an individual having a disease or disorderdependent or modulated by angiogenesis, wherein the disease or disordercan be treated by the inhibition of angiogenesis is disclosed.Generally, the method comprises administering to the tissue acomposition comprising an angiogenesis-inhibiting amount of Dearinhibitor.

In a related embodiment, the methods of the present invention providefor a method of inhibiting angiogenesis in a tissue of an individual atrisk for developing an angiogenic disease or disorder.

Where the growth of new blood vessels is the cause of, or contributesto, the pathology associated with a disease, inhibition of angiogenesiswill reduce the deleterious effects of the disease. Examples includetumors, rheumatoid arthritis, diabetic retinopathy, inflammatorydiseases, restenosis, and the like. Where the growth of new bloodvessels is required to support growth of a deleterious tissue,inhibition of angiogenesis will reduce the blood supply to the tissueand thereby contribute to reduction in tissue mass based on blood supplyrequirements. Examples include growth of tumors where neovascularizationis a continual requirement in order that the tumor growth beyond a fewmillimeters in thickness, and for the establishment of solid tumormetastases. Another example is coronary plaque enlargement.

The invention provides for a method for the inhibition of angiogenesisin a tissue, and thereby inhibiting events in the tissue which dependupon angiogenesis.

The treatment will involve the administration of a Dear inhibitor. Thetreatment may involve a combination of treatments, including, but notlimited to a Dear inhibitor in combination with other angiogenicinhibitors, chemotherapy, radiation, surgery, or other treatments knownto those of skill in the art to inhibit angiogenesis. Examples ofangiogenic inhibitors that may be used in combination with the Dearinhibitor of the present invention are: direct angiogenesis inhibitors,Angiostatin, Bevacizumab (Avastin), Arresten, Canstatin, Combretastatin,Endostatin, NM-3, Thrombospondin, Tumstatin, 2-methoxyestradiol, andVitaxin; and indirect angiogenesis inhibitors: ZD1839 (Iressa), ZD6474,OS1774 (Tarceva), CI1033, PKI1666, IMC225 (Erbitux), PTK787, SU6668,SU11248, Herceptin, and IFN-α, CELEBREX® (Celecoxib), THALOMID®(Thalidomide), and IFN-α.

Thus, in connection with the administration of a Dear inhibitor, acompound which inhibits angiogenesis indicates that administration in aclinically appropriate manner results in a beneficial effect for atleast a statistically significant fraction of patients, such asimprovement of symptoms, a cure, a reduction in disease load, reductionin tumor mass or cell numbers, extension of life, improvement in qualityof life, or other effect generally recognized as positive by medicaldoctors familiar with treating the particular type of disease orcondition.

Examples of Dear inhibitors include, but are not limited to, moleculeswhich block the binding of AngII, ET-1 and/or other ET-1 or AngII-likeligands to Dear, compounds which interfere with downstream signalingevents of Dear, or other compounds or agents that inhibit activation ofthe receptor. Such compounds include antibodies that bind to Dear andprevent binding of AngII, ET-1 or other mimetic ligands. Preferably, theantibody is a humanized antibody. Preferably, the antibody is a singlechain antibody or F(ab)² fragment. Other inhibitors including smallmolecules that bind to the Dear domain that binds to ET-1, soluble Dearreceptors, peptides containing the Dear ET-1 and/or AngII bindingdomains, etc.

There are a variety of diseases or disorders in which angiogenesis isbelieved to lead to negative consequences, referred to as pathologicalangiogenesis, including but not limited to, inflammatory disorders suchas immune and non-immune inflammation, chronic articular rheumatism andpsoriasis, disorders associated with inappropriate or inopportuneinvasion of vessels such as diabetic retinopathy, neovascular glaucoma,restenosis, capillary proliferation in atherosclerotic plaques andosteoporosis, and cancer associated disorders, such as solid tumors,solid tumor metastases, angiofibromas, retrolental fibroplasia,hemangiomas, Kaposi sarcoma and the like cancers which requireneovascularization to support tumor growth. In a preferred embodiment ofthe present invention, the methods are directed to inhibitingangiogenesis in a mammal with cancer.

As described herein, any of a variety of tissues, or organs comprised oforganized tissues, can support angiogenesis in disease conditionsincluding skin, muscle, gut, connective tissue, joints, bones and thelike tissue in which blood vessels can invade upon angiogenic stimuli.

The individual treated in the present invention in its many embodimentsis desirably a human patient, although it is to be understood that theprinciples of the invention indicate that the invention is effectivewith respect to all mammals, which are intended to be included in theterm “patient”. In this context, a mammal is understood to include anymammalian species in which treatment of diseases associated withangiogenesis is desirable, particularly agricultural and domesticmammalian species.

In a preferred embodiment, the present invention is directed to methodsof inhibiting angiogenesis in a tissue of a mammal having pathologicalangiogenesis as in cancer, and in particular breast cancer and theadministration of the Dear inhibitor eliminates or reduces the presenceof the cancer.

B. Enhancing Angiogenesis

In an alternative embodiment of the present invention, Dear activatorsare used to stimulate angiogenesis in tissues or organs in need ofneovascularization or additional blood supply. In these instances,delivery of a Dear activator alone or in combination with otherangiogenesis stimulators may be beneficial.

Any condition that would benefit from increased blood flow areencompassed such as, for example, gangrene, diabetes, poor circulation,arteriosclerosis, atherosclerosis, coronary artery disease, myocardialischemia, myocardial infarction, aortic aneurysm, arterial disease ofthe lower extremities, cerebrovascular disease, etc. In this manner, themethods of the invention may be used to treat peripheral vasculardiseases by administering Dear activators to promote vascularization.Likewise, the Dear activators are useful to treat a diseased or hypoxicheart, particularly where vessels to the heart are partially orcompletely obstructed. Other organs with arterial sclerosis may benefitfrom Dear activation Likewise, organs whose function may be enhanced byhigher vascularization may be improved by an activation of Dear. Thisincludes kidneys or other organs which need an improvement in function.In the same manner, other disorders which could benefit from increasedblood flow include ischemic bowel disease, cerebro vascular disease,impotence of a vascular basis, and the like. Additionally, formation ofnew blood vessels in the heart is critically important in protecting themyocardium from the consequences of coronary obstruction. Administrationof a Dear activator into ischemic myocardium can enhance the developmentof collaterals, accelerate the healing of necrotic tissue and preventinfarct expansion and cardiac failure.

Additionally, Dear activators are useful to prepare a transplant sitefor tissues or organs of interest by increasing vascularization. Suchorgan transplants include, but are not limited to, pancreas, kidney,heart, lung, liver, etc. Dear activators may also be used in combinationwith other implants as a surgical adhesion barrier.

Following in vitro fertilization, the embryo is implanted in a femalefor gestation. The methods of the invention can be used to prepare theuterine vascularized bed for embryo implantation. In this embodiment,Dear activators are introduced prior to implantation so as to promoteblood vessel formation in the uterine wall prior to implantation of theembryo, thus promoting fetal-maternal vascular plexus. Likewise, Dearactivators can also enhance fetal-maternal vascular plexus formation,and/or robust placental vasculature for successful pregnancy/gestationof both natural and in vitro fertilized embryos.

Skilled artisans are able to determine when therapy is beneficial andwhere therapy is contraindicated. In general, patients with known tumorsor pathological angiogenesis should not be given the Dear activators ofthe present invention.

III. Tumor Pro-Malignant Potential Decreasing Pro-Malignant Potential ofTumors

In another embodiment of the present invention, a method of decreasingthe pro-malignant potential of a tumor is disclosed. Generally, themethod consists of administering to the tumor, systemically or locally,a composition comprising a Dear inhibitor at a dose which decreasespro-malignant parameters such as, but not all inclusive, nuclearpleomorphism, nuclear hyperchromasia, vascular invasion, mosaic tumorvessels, chaotic tumor vessels, tumor metastasis, etc.

Formulations

The Dear activators and inhibitors of the present invention may beadministered to an individual via intravenous (I.V.), intramuscular(I.M.), subcutaneous (S.C.), intradermal (I.D.), intraperitoneal (I.P.),intrathecal (I.T.), intrapleural, intrauterine, rectal, vaginal,topical, intratumor and the like. The Dear modulators (either activatorsor inhibitors) can be administered parenterally by injection or bygradual infusion over time and can be delivered by peristaltic means.

Administration may be by transmucosal or transdermal means. Fortransmucosal or transdermal administration, penetrants appropriate tothe barrier to be permeated are used in the formulation. Such penetrantsare generally known in the art, and include, for example, fortransmucosal administration bile salts and fusidic acid derivatives. Inaddition, detergents may be used to facilitate permeation. Transmucosaladministration may be through nasal sprays, for example, or usingsuppositories. For oral administration, the compounds of the inventionare formulated into conventional oral administration forms such ascapsules, tablets and tonics.

For topical administration, the pharmaceutical composition (inhibitor oractivator of Dear activity) is formulated into ointments, salves, gels,or creams, as is generally known in the art.

The activators and inhibitors of Dear are conventionally administeredintravenously, as by injection of a unit dose, for example. The term“unit dose” when used in reference to a therapeutic composition refersto physically discrete units suitable as unitary dosage for the subject,each unit containing a predetermined quantity of active materialcalculated to produce the desired therapeutic effect in association withthe required diluent; i.e., carrier, or vehicle.

The compositions are administered in a manner compatible with the dosageformulation, and in a therapeutically effective amount. The quantity tobe administered and timing depends on the subject to be treated,capacity of the subject's system to utilize the active ingredient, anddegree of therapeutic effect desired. Precise amounts of activeingredient required to be administered depend on the judgment of thepractitioner and are peculiar to each individual.

The Dear activators and inhibitors useful for practicing the methods ofthe present invention are of any formulation or drug delivery systemcontaining the active ingredients, which is suitable for the intendeduse, as are generally known to those of skill in the art. Suitablepharmaceutically acceptable carriers for oral, rectal, topical orparenteral (including inhaled, subcutaneous, intraperitoneal,intramuscular and intravenous) administration are known to those ofskill in the art. The carrier must be pharmaceutically acceptable in thesense of being compatible with the other ingredients of the formulationand not deleterious to the recipient thereof.

As used herein, the terms “pharmaceutically acceptable”,“physiologically tolerable” and grammatical variations thereof, as theyrefer to compositions, carriers, diluents and reagents, are usedinterchangeably and represent that the materials are capable ofadministration to or upon a mammal without the production of undesirablephysiological effects.

Formulations suitable for parenteral administration conveniently includesterile aqueous preparation of the active compound which is preferablyisotonic with the blood of the recipient. Thus, such formulations mayconveniently contain distilled water, 5% dextrose in distilled water orsaline. Useful formulations also include concentrated solutions orsolids containing the compound which upon dilution with an appropriatesolvent give a solution suitable for parental administration above.

For enteral administration, a compound can be incorporated into an inertcarrier in discrete units such as capsules, cachets, tablets orlozenges, each containing a predetermined amount of the active compound;as a powder or granules; or a suspension or solution in an aqueousliquid or non-aqueous liquid, e.g., a syrup, an elixir, an emulsion or adraught. Suitable carriers may be starches or sugars and includelubricants, flavorings, binders, and other materials of the same nature.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active compound in a free-flowingform, e.g., a powder or granules, optionally mixed with accessoryingredients, e.g., binders, lubricants, inert diluents, surface activeor dispersing agents. Molded tablets may be made by molding in asuitable machine, a mixture of the powdered active compound with anysuitable carrier.

A syrup or suspension may be made by adding the active compound to aconcentrated, aqueous solution of a sugar, e.g., sucrose, to which mayalso be added any accessory ingredients. Such accessory ingredients mayinclude flavoring, an agent to retard crystallization of the sugar or anagent to increase the solubility of any other ingredient, e.g., as apolyhydric alcohol, for example, glycerol or sorbitol.

Formulations for rectal administration may be presented as a suppositorywith a conventional carrier, e.g., cocoa butter or Witepsol S55(trademark of Dynamite Nobel Chemical, Germany), for a suppository base.

Formulations for oral administration may be presented with an enhancer.Orally-acceptable absorption enhancers include surfactants such assodium lauryl sulfate, palmitoyl carnitine, Laureth-9,phosphatidylcholine, cyclodextrin and derivatives thereof; bile saltssuch as sodium deoxycholate, sodium taurocholate, sodium glycochlate,and sodium fusidate; chelating agents including EDTA, citric acid andsalicylates; and fatty acids (e.g., oleic acid, lauric acid,acylcarnitines, mono- and diglycerides). Other oral absorption enhancersinclude benzalkonium chloride, benzethonium chloride, CHAPS(3-(3-cholamidopropyl)-dimethylammonio-1-propanesulfonate),Big-CHAPS(N,N-bis(3-D-gluconamidopropyl)-cholamide), chlorobutanol,octoxynol-9, benzyl alcohol, phenols, cresols, and alkyl alcohols. Anespecially preferred oral absorption enhancer for the present inventionis sodium lauryl sulfate.

Alternatively, the compound may be administered in liposomes ormicrospheres (or microparticles). Methods for preparing liposomes andmicrospheres for administration to a patient are well known to those ofskill in the art. U.S. Pat. No. 4,789,734, the contents of which arehereby incorporated by reference, describes methods for encapsulatingbiological materials in liposomes. Essentially, the material isdissolved in an aqueous solution, the appropriate phospholipids andlipids added, along with surfactants if required, and the materialdialyzed or sonicated, as necessary. A review of known methods isprovided by G. Gregoriadis, Chapter 14, “Liposomes,” Drug Carriers inBiology and Medicine, pp. 287-341 (Academic Press, 1979).

Microspheres formed of polymers or proteins are well known to thoseskilled in the art, and can be tailored for passage through thegastrointestinal tract directly into the blood stream. Alternatively,the compound can be incorporated and the microspheres, or composite ofmicrospheres, implanted for slow release over a period of time rangingfrom days to months. See, for example, U.S. Pat. Nos. 4,906,474,4,925,673 and 3,625,214, and Jein, TIPS19:155-157 (1998), the contentsof which are hereby incorporated by reference.

In one embodiment, the Dear activator or inhibitor of the presentinvention can be formulated into a liposome, microparticle,nanoparticle, etc. which is suitably sized to lodge in capillary bedsfollowing intravenous administration. When the liposome, microparticleor nanoparticle is lodged in the capillary beds surrounding ischemictissue, the agents can be administered locally to the site at which theycan be most effective. Suitable liposomes for targeting ischemic tissueare generally less than about 200 nanometers and are also typicallyunilamellar vesicles, as disclosed, for example, in U.S. Pat. No.5,593,688 to Baldeschweiler, entitled “Liposomal targeting of ischemictissue,” the contents of which are hereby incorporated by reference.

Preferred particles are those prepared from biodegradable polymers, suchas polyglycolide, polylactide and copolymers thereof. Those of skill inthe art can readily determine an appropriate carrier system depending onvarious factors, including the desired rate of drug release and thedesired dosage.

In one embodiment, the formulations are administered via catheterdirectly to the inside of blood vessels. The administration can occur,for example, through holes in the catheter. In those embodiments whereinthe active compounds have a relatively long half life (on the order of 1day to a week or more), the formulations can be included inbiodegradable polymeric hydrogels, such as those disclosed in U.S. Pat.No. 5,410,016 to Hubbell et al. These polymeric hydrogels can bedelivered to the inside of a tissue lumen and the active compoundsreleased over time as the polymer degrades. If desirable, the polymerichydrogels can have microparticles or liposomes which include the activecompound dispersed therein, providing another mechanism for thecontrolled release of the active compounds.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.All methods include the step of bringing the active compound intoassociation with a carrier which constitutes one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing the active compound into association with a liquidcarrier or a finely divided solid carrier and then, if necessary,shaping the product into desired unit dosage form.

The formulations may further include one or more optional accessoryingredient(s) utilized in the art of pharmaceutical formulations, e.g.,diluents, buffers, flavoring agents, binders, surface active agents,thickeners, lubricants, suspending agents, preservatives (includingantioxidants) and the like.

Compounds of the present methods may be presented for administration tothe respiratory tract as a snuff or an aerosol or solution for anebulizer, or as a microfine powder for insufflation, alone or incombination with an inert carrier such as lactose. In such a case theparticles of active compound suitably have diameters of less than 50microns, preferably less than 10 microns, more preferably between 2 and5 microns.

Generally for nasal administration a mildly acid pH will be preferred.Preferably the compositions of the invention have a pH of from about 3to 5, more preferably from about 3.5 to about 3.9 and most preferably3.7. Adjustment of the pH is achieved by addition of an appropriateacid, such as hydrochloric acid.

The preparation of a pharmacological composition that contains activeingredients dissolved or dispersed therein is well understood in the artand need not be limited based on formulation. Typically suchcompositions are prepared as injectables either as liquid solutions orsuspensions, however, solid forms suitable for solution, or suspensions,in liquid prior to use can also be prepared. The preparation can also beemulsified.

The following is the sequence for rat Dear:

Rattus norvegicus dual endothelin 1, angiotensin II receptor (Dear),mRNA:

GeneID: 446170 Locus tag: RGD:1303105 (SEQ. ID. NO. 1) 1tttctaaatg attacttttc tagatacctg tttacaaaac agaagatcct ccctttgaaa 61ccaaactaaa ctacacttga agaatataaa gtgcacaaag gaagaccacg atgaatcagt 121accacccatc ttcccagcat tcaagaatgt tcagcgcaca ggaggtgcac agtaagtgtt 181cctgagagga gtggatacaa cactcaatta tctgggcatg taatgctgat ctgcggtttc 241ctttacatca gccggcctcc ttcccggttg ggatcaagga agtgaacaga tgcagactca 301ctctggcagg caccactgag ccagccattt actctcactg catagcaaag ttattttgtc 361aacttgtttc caggatcctc tgcttccaca gagcagaaac acctcgtcta ggggattctg 421atccttaccc tcttctttac atttctctct ccagagaagg ttatcctcag ccaaaatcct 481ccagtatcga cacgtctgag ccgcttgcag caggtctttg ggttccagga atgaaagtac 541atagagtgcc agctatggaa aaggaaataa gggaggcaca ctcagacaca tcacagaaga 601aaggttactc tacgaagggt gagcattgag ctgggactgc tggatttcag tgggctgaac 661gaatcaagag aagcagcatt ttaagagcaa agaaaatgct catcattctc acattaggaa 721tctgaggctt tactctgggt aacctgtgat tctattggta tttccttaca aagtgagaac 781aatgccactt atcacaagtt tcttgtgtga gcccagtgct caagctctta gataatccaa 841ataaatgttg ataaagagac tttatattgt tcataccaat tatcaaaaaa tacaagtaca 901tttcatgtca gtgtggtaat aatgttttaa ataacacact tccctacagg gttaagtcta 961tgccattatt cttccacgca gacataaagc acttcccaaa tgaagaacac cccagtagtc 1021agaaacaaaa accatagctg atatgctaag acagggctct ctcctttgta acttcttttt 1081tcctcaggaa gtctttgaaa gaacacagaa gccaagagaa tcttttgggg ttttaccttt 1141tattaatcat ctgtgcttac ttcttaaaat tctaaaacac tttcaaattt gggggactgg 1201tgatggctca gtcagtaagg tttcatgaag atgagggctc ggatcctggc agtcttggaa 1261agtcaggcat ggcagttcta gctatagtca ctgctcactg gacagccagc caccgtagct 1321aaaggcgtaa gctccagact cagtgaaaga cgacatggca aaaacaacat ggatcagctg 1381agggatacac ctctggcctc cacgggcaca tacgtgcagg agcatctgaa tgtacttatg 1441tatacccaca cgaatacata cacatcctac acacatacac actctacagg aggtatcggg 1501catgtaagat aatccagacg aatattcact tcacgccctg atggcagcaa agggatctcg 1561tgttactttc ataagtttag tcaaagagtt ctgatgtaga aaaagctcac aagagcaaac 1621acttttcttc tgggacactg tcacctttaa aaagtactca aaagggggga aagtgccagg 1681aaaaagatga tttatcaatt tgctttcccc cagaattata ttttaattca tcaattttac 1741tcaaatctaa tgccagattc taactaggac tatatttaat gccactagga ctttagagtg 1801atcatctaag aaaggagaaa gcaagactct tcctgttcaa atgaggtttc gggatcatct 1861gtatgaagga tgtggtagtt ttttgatgct gtctttttaa ctgctattta taacatgtgt 1921atagtaattt gagaaaatat ggactatggg gcattatcta atatcacatt atttcttcct 1981tttgataaaa attaagctat gaagtctaat gtcaatatgt gcattatatt taaaccatca 2041gccacacatg gctgtatgac taagtgccta agaatccaat ttttttgtgg tatctctctc 2101tctccctctc tccgtatgtg tgtgtctttc tctgtctctg tctctctctc tctttctctc 2161tgacgaagga tgataagtag aaatgccata aaaacatata gataaatttt atatattggg 2221ggctggagag atggctcagt ggttgagaac actgactgtt cttcagaggt cctgagttaa 2281attcccagca accacatggt agctcacaac catctatatt gcatctgatg ccctcttctg 2341gtgtgtctaa agacagctac cggtatactt acatataata aataaatctt taaaaaaatt 2401ttttatatat taaaaaaaaa tcacataatg taataaccag gagaaatacg aacaatcgat 2461aaaattactg gtcttgaagg ggcattaaat aattagcaaa ataaaaacaa aattaatatt 2521gttgcttagt gaatccagaa ttttgaaaac atccactata tataataaac ataccaacta 2581actaaagtca gcctttagat aacccaggga aaactgaaga gactcggcga cttcacatga 2641agccttactt tatccaagcg gaagaaagca gcaccttggt atgagcacac tttatgtaac 2701agctgtacca aaaagccaca gcagtttgcc aaagtgtcaa gccatgatga gcaggacact 2761gcttacaggc atggctatgt atctggacag cagccatgcg ggtgctgcat ccatgcaggt 2821gagctggccg cccttactca cctctttggg gagcaaggag atgaagtctc gctggaactg 2881gggctcgatc acttgcatca tgtgcttcac ttgtgtgggt tcacagctat cgatgagctc 2941atctaaggcc agcaacttct ctggtccact ccagctctac caaagaggaa ttggacacat 3001tacaaatcca tacagaagac caccagcacc tgcatggcca tgttcgagca gtggaactac 3061atgaaagggg accgtggaca gagaccttgt ctccagaagc caccagagcg atagcagttt 3121ttagtttcag caagtttact cagtaccttt cccgcaaagc attaaaagtc atgactggca 3181gaaaaataag tctgcattta tttttaatta taagacttat gctaacacca agacactggg 3241agacacacaa tatccatctg ggttattgac tag (SEQ. ID. NO. 2) Translation =“MSTLYVTAVPKSHSSLPKCQAMMSRTLLTGMAMYLDSSHAGAASMQVSWPPLLTSLGSKEMKSRWNWGSITCIMCFTCVGSQLSMSSSKASNFSGPLQLYQRGIGHITNPYRRPPAPAWPCSSSGTT”

Example 1

The inbred Dahl/JR rat model is an established model of human essentialhypertension comprised of a salt-sensitive, hypertensive strain (Dahl S)with its cognate salt-resistant, normotensive control strain (Dahl R)(52). To investigate the involvement of Dear in hypertensionpathogenesis we obtained cDNAs spanning the entire amino acid codingregion for both Dahl S and Dahl R receptors. Two nucleotide differenceswere detected resulting in two non-conservative amino acidsubstitutions: T²⁸¹⁴ (Dahl S)/C²⁸¹⁴ (Dahl R) nucleotide transitionresulting in S44P substitution and T²⁹⁰¹ (Dahl S)/C²⁹⁰¹ (Dahl R)nucleotide transition resulting in M74T substitution (FIG. 1A, 1B). TheS44P substitution is located in the putative AngII binding site in theextracellular domain; while the M74T substitution is located in theputative transmembrane domain (FIG. 1B). The Dahl S cDNA nucleotidesequence is identical to the previously reported Sprague Dawley ratbrain Dear cDNA (1). We note that both the Dahl S and Dahl R rat strainswere derived from the Sprague Dawley strain (52).

The S44P substitution spans the predicted AngII binding domain withinthe Dahl R Dear variant (1) (FIG. 1B) suggesting the hypothesis thatAngII-binding will most likely be different between Dahl S and Dahl Rreceptors. In order to examine this hypothesis, we first determined thatthere is no significant difference in Dear expression levels betweenDahl S and Dahl R rats at 12 weeks of age in both male and female ratsas detected by western blot analysis comparing Dahl S and Dahl R kidneymembranes (FIG. 1C). To examine hormone binding, both Dahl S and Dahl Rreceptors were transiently expressed in Cos1 cells respectively andtested for both AngII and ET-1 binding. Dahl R Dear do not exhibit AngIIbinding, but exhibit normal ET-1 binding as shown by direct radioligandbinding (FIG. 2 and Table 1), and by west-western blot analysis (i.e.,labeled ligand [west] binding to receptor polypeptide on western blot)of Dahl S and Dahl R rat kidney membranes and Dahl S and Dahl R DearCost-transfectant cell membranes (FIG. 2C). These data demonstrate thatthe Dahl S Dear variant is a dual receptor binding both ET-1 and AngIIsimilar to the brain-derived clone first characterized (1), but that theDahl R Dear variant responds solely to ET-1 stimulation. Two affinitybinding sites for ET-1 are detected in Dahl S and Dahl R receptors (FIG.3, Table 1) and two affinity binding sites for AngII in Dahl S receptors(FIG. 3, Table 1) consistent with previous characterization (1).Furthermore, when compared to the Dahl R Dear S44P/M74S variant, theDahl S Dear S44/M74 variant exhibits 3-fold increased affinity for ET-1(Dahl R: S44P/M74T K_(H) ET-1=12.0±1.12 pM; Dahl S: S44/M74 K_(H)ET-1=4.42±0.89 pM, P<0.001, Table 1)—suggesting an enhanced response ofthe Dahl S receptor to ET-1 stimulation compared to the Dahl R receptor.

Based on its localization to the predicted AngII-binding site, it islikely that the S44P substitution accounts for the observed absent AngIIbinding in Dahl R Dear. Interestingly, this S44P substitution andresultant differential AngII binding elucidates for the first time anatural occurring mutation within a peptide-ligand binding domainpredicted by the molecular recognition theory (1).

Having found functionally significant variants between Dahl S and Dahl RDear genes, we then investigated the potential genetic contribution tohypertension susceptibility by performing independent QTL analysis onboth male (n=106) and female (n=102) F2 [RxS]-intercross rats phenotypedfor blood pressure by radiotelemetry after 8 and 12 weeks of high salt(8% NaCl) challenge respectively (Table 2). The high-salt diet challengewas extended 4 weeks longer for the F2 [RxS]-intercross female ratssince female BP phenotype was much lower than in male F2[RxS]-intercross rats (average F2 [RxS]-intercross male SBP after eightweeks of high salt diet=157.2±14.2 mmHg; average F2 [RxS]-intercrossfemale SBP after twelve weeks of high salt diet=145.0±11.4 mmHg, Table2). Using an SSCP-based Dear gene-specific marker (FIG. 4A), we mappedthe rat Dear to chromosome 2 (physical position in the current assemblyof the rat genome: 2q34, 176.687 Mb), 4.5 cM centromeric to theα1-Na,K-ATPase locus, ATP1A1. A total chromosome-2 scan was then donewith 11 informative markers that distinguish Dahl R and Dahl S strains.Marker regression and interval mapping analyses detect a singlechromosomal region with suggestive linkage to mean, systolic anddiastolic BP, peaking at ATP1A1+2 cM (LOD=1.70) in the F2 [R×S] malecohort (FIG. 4B, Table 2). In contrast, chromosome-2 scan analysis of F2[R×S]-intercross females revealed two QTLs on chromosome 2, one centeredat D2Rat143 (LOD=2.43, significant linkage) and the other centered atDear−5 cM (LOD=3.61, highly significant linkage) (FIG. 4C, Table 2). Toassess pathophysiological relevance, analysis of Dear allele-specificcontribution (Table 3) reveals that the Dahl S S44/M74 variant increasessusceptibility to hypertension regardless of BP parameter—systolic,diastolic or mean arterial pressure with greatest changes in meanarterial pressure (ANOVA P<10⁻³ to 10⁻⁴). Concordance of results indifferent blood pressure parameters provide evidence delineating theDear locus as a gene for hypertension susceptibility in F2[RxS]-intercross female rats.

To date, only Dear (shown here) and ATP1A1 (38, 43) genes exhibitfunctionally significant variants between Dahl S and Dahl R rats withdemonstrated pathogenic relevance to hypertension. These data forwardsaid two loci as candidate genes for the Dear−5 cM QTL region onchromosome 2 affecting BP in female F2 [R×S]-intercross rats based on a4-parameter analysis framework for hypertension genes (37, 54). Thecausal role of ATP1A1 in hypertension has been shown by transgenesis inboth male and female Dahl S rats (38). While putative gene interactionsneed to be investigated for Dear, ATP1A1-Na,K,2Cl-cotransporter (NKCC2)gene-interaction has been detected to increase susceptibility to highblood pressure in cosegregation analysis of F2 [R×S] intercross rats(39). This epistatic nature of the ATP1A1 effect on BP could account forthe reduced statistical significance of linkage to BP detected at ATP1A1when analyzed as a single locus as done in this chromosome 2 scan and inprevious F2-intercross and congenic studies (34, 53).

To further analyze the Dear locus in the context of previous genetic ratmodel studies which report chromosome 2 QTLs for BP which span the Dearlocus (31, 34, 41, 51, 59), we assessed Dear variants on WKY, SHR, BNand LEW rat strains used in said studies by SSCP analysis. As shown inFIG. 4A, the Dahl R Dear S44P/M74T variant is detected in Dahl R and LEWstrains, while the Dahl S Dear S44/M74 variant is detected in SHR, WKYand BN strains. Detection of variant-specific alleles in the differentstrains was corroborated by direct nucleotide sequencing of twoindependent Dear cDNA clones spanning the entire amino acid codingregion (data not shown). Since SHR, WKY and BN rat strains have theS44/M74 Dahl S Dear allele, the Dear locus is a priori eliminated as acandidate gene for the chromosome 2 QTL for BP in F2-intercross studiesderived from these strains (31, 41, 51, 59). The Dear locus is alsoeliminated as candidate gene for the reported chromosome 2 BP QTL in F2[Dahl S×LEW]-intercross study which investigated males only (34), sincethe Dear S-variant cosegregates with high blood pressure in females.

Thus, observations from genetic, molecular and pathophysiologicalanalyses suggest that modification in the balance of AngII and ET-1receptor systems through variant Dear contributes to hypertensionsusceptibility in female F2 [RxS]-intercross rats. The data reiteratethe importance of gender-specific factors in hypertension susceptibilityand the role of Dear in AngII-ET-1 response-balance.

Materials and Methods

Characterization of Dahl S and Dahl R Dear cDNAs

Dahl S and Dahl R Dear cDNAs were RT-PCR from Dahl S/JRHsd and DahlR/JRHsd rat kidney PolyA⁺ RNAs respectively (Forward primer:5′-AAG-AAA-GCA-GCA-CCT-TGG-T-3′ (SEQ. ID. NO. 3); Reverse primer:5′-CGT-GGA-CAG-AGA-CCT-TGT-CT-3′ (SEQ. ID. NO. 4)) and subsequentlysubcloned into the PT-vector system (Clontech, Palo Alto, Calif.).Primer sequences were obtained from the previously reported SpragueDawley Dear cDNA (GenBank accession number AY664492). The cDNAs (432 bp)encompassing the entire Dear amino acid coding region was then sequencedon both strands. Six Dahl S and six Dahl R rats were sequenced showingno intra-strain sequence heterogeneity.

Detection of the Dear Gene S44P/M74T Variant by Single StrandConformation Polymorphism (SSCP) Analysis

Dahl S/jrHsd, Dahl R/jrHsd, LEW/SsNHsd, WKY/NHsd, SHR/NHsd and BN/Hsdrats were purchased from Harlan Inc. (Indianapolis, Ind.). SSCP analysiswas performed on genomic DNA isolated from the different rat strainsessentially as described (60). The SSCP marker was based on a PCRproduct encompassing nucleotides 2774-2911 (spanning the S44Psubstitution) within the amino acid encoding region of the Dear cDNA(forward primer: 5′-GCT-ATG-TAT-CTG-GAC-AGC-AGC-3′ (SEQ. ID. NO. 5);reverse primer: 5′-AGT-GAA-GCA-CAT-GAT-GCA-AGT-3′(SEQ. ID. NO. 6);product: 137 bp) (1). The SSCP marker was detected by 6% non-denaturingpolyacrylamide gel electrophoresis.

Receptor Expression and Membrane Preparation

The Dahl S and Dahl R Dear cDNAs were subcloned directionally (5′ to 3′)into the pcDNA (+) expression vector (Invitrogen, Carlsbad, Calif.) andtransiently expressed in Cost cells (ATCC). Cos1 cells were transfectedwith the expression vectors via lipofectin-mediated gene transfer andcell membranes were isolated 72 hr post-transfection for hormonebinding. Rat kidney membranes were prepared essentially as described(42). COS-1 cell membranes were isolated as described (36). Briefly,cells were washed twice in phosphate-buffered saline and homogenized in10-fold ice-cold buffer (0.25 M sucrose, 1 mM EDTA, 50 μg/ml aprotinin,10 μg/ml leupeptin, 100 μM phenylmethylsulfonyl fluoride, 25 mMImidazol/HCl, pH 7.4). The homogenate was centrifuged at 5,000 g for 15min and the pellet was discarded. The supernatant was then centrifugedat 27,500 g for 30 min and the resulting pellet was washed twice inice-cold suspension buffer (5 mM MgCl₂, 0.2 mM EDTA, 50 mM Hepes, pH7.4). The final pellet was resuspended into the appropriate assay bufferand quickly frozen in liquid nitrogen. The membrane preparations werestore at −80° C. until use. Protein concentrations of the membranes weredetermined by BCA protein assay kit (PIERCE).

Radioligand Binding Assays

Binding of [¹²⁵I]Tyr⁴-angiotensin II and [¹²⁵I]Tyr¹³-endothelin-1 toCOS-1 membranes was performed by a rapid filtration method (32, 50).Briefly, [¹²⁵I]Tyr⁴-angiotensin II (0.25˜6.5 nM) or[¹²⁵I]Tyr¹³-endothelin-1 (0.045˜1.46 nM) was incubated with membranes(100 μg) for 20 min at 37° C. in 1000 buffer A (5 mM MgCl₂, 0.2 mM EDTA,10 mg/ml BSA, 10 mM Hepes, pH7.4). Binding reactions were terminated bythe addition of 1 ml ice-cold buffer A and immediately filtered througha Whatman GF/C filter (presoaked overnight at 4° C. in 10 mg/ml BSA) andsubsequently washed with 15 ml ice-cold buffer A. Specific binding wasdetermined as the difference between the total radioactivity bound tomembranes and the radioactivity bound to blanks containing 1 μM AngII or1 μM ET-1. The dissociation constant (K_(d)) and maximum ligand-bindingsites (B_(max)) were determined using Hill plot analysis (55). Hillcoefficient values (h) were calculated from the relationship ln[B/(B_(max)−B)]=h ln [free Radioligand]−ln K_(d). An F test (P<0.05) wasused to determine whether the saturation binding curves best fitted oneor two independent binding sites. The data were best fit by two affinitystates determined by Scatchard plot analysis; K_(H) and K_(L) designatethe K_(d) for high- and low-affinity states of the receptor,respectively (44). Most results are expressed as the mean±SE (standarderror) from three to five independent experiments.

Western and West-Western Blotting Analysis

A polyclonal rabbit antipeptide antibody raised against the syntheticpeptide P₅₁LLTSLGSKE₆₀ (SEQ. ID. NO. 7) was utilized for western blotanalysis (1). Plasma membranes (40 μg protein/lane) were subjected to12.5% SDS-PAGE and the separated proteins electro-transferred onto PVDFmembranes which were incubated with blocking buffer (0.3% Tween-20, 5%non-fat milk, 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na₂HPO₄, and 1.5 mMKH₂PO₄, pH7.4) for 2 hr at room temperature, and then incubated withprimary antibody (1:500) for 16 hr at 4° C. The PVDF membranes were thensequentially incubated with biotinylated goat anti-rabbit IgG followedby immunostaining with horseradish peroxidase-linked streptavidin. Toconfirm the interaction between Dear and ligands, we performedwest-western blot analysis (62). Briefly, protein blots of kidney andCOS-1 cell membranes were incubated with radioligands (0.5 μCi in 10 ml)in buffer A at 37° C. for 16 hr. PVDF membranes were then washed threetimes for 15 min with buffer A at 37° C. and exposed to X-ray film at−80° C. for 1-3 days.

Genetic Crosses

Dahl S/jrHsd and Dahl R/jrHsd rat strains (Harlan, Indianapolis, Ind.)were used to develop the F2 cohort. The F2 cohort was derived frombrother-to-sister mating of F1 (R female×S male) hybrids to produce theF2 male (n=106, carrying exclusively Y chromosomes from the Dahl Sgenetic background) and F2 female (n=102) segregating populations.

Phenotypic Characterization of F2 Cohorts

All animal procedures were performed in accordance with institutionalguidelines. Animals were maintained on a LabDiet 5001 rodent chow(Harlan Teklad, Madison Wis.) containing 0.4% NaCl from weaning untilthe high salt diet begun at 12 weeks of age. The food pellets and waterwere made available ad lib. Blood pressure (BP) was measured essentiallyas described (38) using intra-aortic abdominal radiotelemetric implants(DATASCIENCE) obtaining non-stressed blood pressure measurements takingthe average over ten-seconds every 5 minutes for 24 hours (38). Systolic(SBP), diastolic (DBP) and mean arterial pressures (MAP) were obtainedalong with heart rate and activity. The protocol for the F2 rats was asfollows: implant surgery at 10 weeks of age; only rats with nopost-operative complications were used; after 12 days, baseline BPlevels were obtained. High salt (8% NaCl) challenge was begun at 12weeks of age and maintained for eight weeks for male and twelve weeksfor female F2-intercross rats; a longer high-salt challenge wasnecessary for females to attain a similar F2-mean BP since BP in femalesis lower. BP values used for phenotype are the averages obtained in thefinal week of the salt loading from a 24-hour recording during ano-entry day ascertaining non-stress BP. We note that baseline BP meansfor SBP, DBP and MAP were equivalent, ±1 mmHg range for all three BPparameters among the different Dear genotypes (P>0.5).

Intercross Linkage Analysis

Genotyping was done with 10 chromosome-2 microsatellite markersinformative for our Dahl [R×S] intercross and one SSCP-based Dear marker(described above). Marker regression and QTL analyses was performed withthe Map Manager QTXb17 (MMQTXb17) program (46) using MAP as quantitativetrait. MMQTXb17 generates a likelihood ratio statistic (LRS) as ameasure of the significance of a possible QTL. Genetic distances werecalculated using Kosambi mapping function (genetic distances areexpressed in cM). Critical significance values (LRS values) for linkagewere determined by a permutation test (2000 permutations at 10 cMinterval) on our male and female progenies using Kosambi mappingfunction and a free regression model. Thus, the minimum LRS values forthe F2 male cohort were for Suggestive linkage=4.1 (LOD=0.89); forSignificant linkage=10.6 (LOD=2.30); for Highly Significant linkage=18.4(LOD=4.00) and for the F2 female cohort were for Suggestive linkage=3.9(LOD=0.85); for Significant linkage=9.9 (LOD=2.15); for HighlySignificant linkage=16.6 (LOD=3.61). LRS 4.6 delineates LOD 1-supportinterval. Confidence interval for a QTL location was estimated bybootstrap resampling method wherein histogram single peak delineates theQTL and peak widths define confidence interval for the QTL. Histogramswhich show more than one peak warn that the position for the QTL is notwell defined or that there may be multiple linked QTLs (QTX Map Manager)(46).

Example 2

We isolated the mouse Dear gene from a 129 SVJ mouse genomic library.Molecular characterization detects a one-exon transcription unit (FIG.5A). The mouse receptor polypeptide contains 127 amino acids showing 78percent homology with the rat receptor and binds solely ET-1 (data notshown) resembling the recently characterized Dahl R Dear S44P/M74T ratvariant (2), and suggesting that observations in Dear^(−/−) mice aremost likely not AngII-mediated. The mouse Dear mRNA is detected in alltissues tested with the highest level of expression in kidney and aorta(FIG. 5B).

Targeted inactivation of Dear^(−/−) in ES cells was done by replacementof the genomic region spanning amino acids 81-127 of Dear with aPGK-neomycin cassette (FIG. 5A) resulting in the deletion of the last 47amino acids of the Dear polypeptide, including the putative G-proteininteracting domain (1). Screening for homologous recombination was doneon 196 G418-resistant colonies by Southern blot analysis (FIG. 5C).Targeting events were confirmed by polymerase chain reaction (PCR)amplification using a neomycin-specific primer and a primer flanking theintegration site. Production of the expected size fragment (5.5 Kb) wasindicative of a targeting event (FIG. 5D). Five ES cell clones carryingthe targeted Dear mutation were injected into 129SVJ blastocysts andimplanted into pseudopregnant foster mothers. We obtained 14 chimericmice (representative of 2 independent targeted ES cell clones). Chimeraswere bred to C57BL/6J mice and shown to germ line transmit, producing F1progeny.

Heterozygous Dear-deficient (Dear^(+/−)) male and female mice(backcross-10 inbred C57BL/6 mouse strain) exhibit 50% less Dear proteinin mouse kidney protein blot analysis as detected using an anti-mouseDear anti-peptide specific antibody (FIG. 5F; P<0.05, t-test), less bodyweight at 5 and 6 months of age (males: P=0.007; females: P=0.0006)(FIG. 5I) and decreased blood pressure in female (Dear^(+/+): 134±4.0mmHg vs Dear^(+/−): 112.3±6.1; P<0.01) but not in male mice while heartrate remains equivalent (FIG. 5G-H). Gender-specific blood pressureeffects are concordant with observations in a recent study of ratgenetic hypertension wherein Dear variants cosegregated withhypertension in female but not in male F2-intercross rats (2).

To assess potential embryonic lethality of the gene-targeting event, weanalyzed progeny from F1-Dear^(+/−) male and female cross which weregenotyped for the presence/absence of the targeted Dear allele.Sixty-eight pups were produced from 17 liters showing the followinggenotype distribution: 29 wild type (+/+), 39 heterozygous (+/−), 0 null(−/−). The absence of null genotypes in the F2 progeny demonstrates thatDear null mutation is embryonic lethal.

In order to investigate embryonic lethality in Dear^(−/−) embryos, weanalyzed embryos from timed-pregnancies derived from Dear^(+/−) mice atdifferent stages of development to determine the exact developmentalstage in which lethality occurs. We produced 129 embryos (from E9.5 toE12.5) detecting 33 (−/−), 67 (+/−) and 29 wildtype (+/+) genotypes(FIG. 5D). This conforms to the expected segregation ratio 1 (+/+):2(+/−):1 (−/−) for a standard (+/−)×(+/−) intercross. Analysis of theembryos revealed that lethality occurs around E12.5.

Anatomic analysis of E9.5-E12.5 embryos reveals absent yolk-saccollecting vessels associated with homozygous Dear^(−/−)-deficiency(FIG. 6A-F); heterozygous Dear^(+/−) deficient embryos exhibit normalyolk sac vascularization (data not shown). All embryos with absentyolk-sac collecting vessels are Dear^(−/−) by genotype; heterozygousDear^(+/−) deficient embryos are not distinguishable from Dear embryos(data not shown). Hemorrhagic, resorbed embryos were detected as earlyas E10.5 but mostly at E12.5 (FIG. 6D). Although smaller and strikinglypaler than Dear^(+/+) and Dear^(+/−) embryos, Dear^(−/−) embryos exhibittwo size phenotypes: a dysmorphic phenotype detected at E10.5-E12.5(FIG. 6A-C) that is relatively larger than a second, hypoplasticphenotype (FIG. 6E-F). In order to determine whether genetic variationinfluences the null phenotype, speed congenics were done onto C57BL/6jgenetic background and backcross-10 null mice were generated andanalyzed confirming embryonic range of lethality, absent yolk-saccollecting vessels and both embryo morphology phenotypes in Dear^(−/−)embryos (FIG. 6A-F).

At E10.5 and E11.5, blood-filled pumping hearts were detected in largerdysmorphic Dear^(−/−) mice (FIG. 6B, 6C), but not apparent inhypoplastic Dear^(−/−) mice (FIG. 6E, F). Dear^(−/−) E10.5 embryos lackthe vascular network formation marked by prominent blood-filled dorsalaortae (FIG. 6G) and blood vessels in the cranial region which arenormal features characteristic of E9.5 Dear^(+/+) embryo (FIG. 6G leftpanel). Furthermore, E10.5 Dear^(−/−) embryos exhibit disorganized,blood-filled pools in the cranial region without apparent connection toa blood-filled heart (FIG. 6G) observed to pump (data not shown) despitelack of vascular networking. In contrast to E11.5 Dear^(+/+) embryo,E11.5 dysmorphic-type Dear^(−/−) embryo exhibits minimal and alteredvascular networks in both cranial and caudal regions, dilated heartalbeit blood-filled, and altered brain development (FIG. 6H).Furthermore, while both Dear^(−/−) and Dear^(+/+) E11.5 hearts pump,observation of cardiac pumping reveals single chamber filling andcontraction in Dear^(−/−) embryos, in contrast to distinct filling andpumping of ventricles in E11.5 Dear^(+/+) embryo (data not shown). InE10.5 and E11.5 hypoplastic Dear^(−/−) embryos, minimal cardiovasculardevelopment is detected (FIG. 6E-F). Altered brain and cardiacdevelopment is confirmed in the analysis of cleared, fixed E11.5 embryosrevealing poor delineation of brain regions particularly thetelencephalon and cardiac chamber formation (FIG. 6I).

In summary, analysis of dysmorphic Dear^(−/−) embryos at E10.5 and E11.5detects blood-filled hearts (FIG. 6G-H) which contracted, despiteaberrant vascular formation typified by disorganized, blood-filled poolsin the cranial region without apparent connection to a blood-filledheart (FIG. 6G), or minimal vascular networks in both cranial and caudalregions, and a dilated blood-filled heart (FIG. 6H). This contrasts theprominent vascular network marked by blood-filled dorsal aorta and bloodvessels in the cranial region which are characteristic features of E9.5Dear^(+/+) embryo (FIG. 6G). Furthermore, analysis of fixed E11.5embryos reveals abnormal brain and cardiac development in Dear^(−/−)embryos (FIG. 6I).

Histological analysis of Masson-trichrome stained E10.5-E11.5 embryosections confirm minimal to absent collecting vessels in the yolk sac inDear^(−/−) embryos (FIG. 7A) compared with Dear^(+/+) (FIG. 7B) atE10.5. The yolk-sac plexus of smaller vessels are present, with fewernucleated red blood cells (FIG. 7A). A few are enlarged (FIG. 7A). Bloodislands are present but the number of nucleated red blood cells isdecreased in Dear^(−/−) embryos (FIG. 7A) compared with Dear^(+/+)embryos (FIG. 7B). At E12.5, comparative histological analysis revealsamorphous cellular areas with a lack of apparent organogenesis indysmorphic-type Dear^(−/−) embryos (FIG. 7C) compared with Dear^(+/+)(FIG. 7D). High magnification reveals large areas of nucleated red bloodcells in the ventral midportion that are not contained in blood vesselsor in recognizable liver tissue (FIG. 7C). The heart is thin-walled,enlarged and with poor delineation of chambers in Dear^(−/−) embryos(data not shown) corroborating anatomical observations (FIG. 6). Bloodvessels with few nucleated red blood cells are rudimentary, thin-walledand sparse in Dear^(−/−) (FIG. 7C) compared with Dear^(+/+) (FIG. 7D)embryos most evident in the cranial region. Rudimentary, thin-walledblood vessels are detected in the perineural regions with sparsenucleated rbcs in Dear^(−/−) (FIG. 7E), in contrast to Dear^(+/+)embryos which exhibit perineural blood vessels filled with nucleatedrbcs and with perivascular sheaths (FIG. 7D, F), thus corroborating theobserved vascular network deficiency evident in anatomical analyses(FIG. 6). Concordant with sparse perineural vessels, only a fewpenetrating capillaries are evident in Dear^(−/−) embryo neuroepithelium(FIG. 7G) in contrast to Dear^(+/+) embryo (FIG. 7H). Scatterednucleated rbcs are detected in the neuroepithelium (FIG. 7G) suggestingpossible vascular leakiness and/or failure of vasculogenesis.Immunohistochemical analyses comparing E12.5 Dear^(+/+) and Dear^(−/−)embryos do not detect upregulation of VEGF, VEGF-receptor 2 flk-1, orangiopoietin-1 and -2 expression (data not shown).

To further analyze vascular deficits, immunohistochemical staining forsmooth muscle cell (smc) α-actin reveals scattered expression in E12.5Dear^(−/−) embryos and intense staining in the embryo-placenta vascularconnection (FIG. 9A-F). Perineural blood vessels exhibit a-actinimmunostaining in Dear^(−/−) embryos but have minimalangiogenic-branching in contrast to Dear^(+/+) embryos whereinangiogenic sprouting is evident (FIG. 9A-F). Closer histologicalanalysis reveals sporadic blood islands incompletely circumscribed byα-actin stained single-cell vascular wall in Dear^(−/−) embryos (FIG.9A-F).

To investigate mouse Dear temporal and spatial expression patterns, weanalyzed Dear expression in E9.5-E12.5 wild type embryos using ananti-mouse Dear specific anti-peptide antibody validated to detect Dearpolypeptide (FIG. 10). At E9.5 days, we detect Dear expressionpredominantly in the heart, yolk sac mesodermal layer and endothelium,fetal vascular endothelium in the placenta, dorsal aorta, and ependymallayer of the neural tube (FIG. 10A). These expression patterns persistat E12.5 days, wherein we detect increased expression in somehemangioblasts in yolk sac blood islands (FIG. 10B), as well as moreprominent expression in the ependymal layer of the neuroepithelium andin perineural blood vessel walls (FIG. 10B). Observed temporal andspatial expression patterns are concordant with vascular, cardiac andneuroepithelial phenotypes observed in Dear^(−/−) deficient mice.

In contrast to minimal blood islands in the yolk sac, we detectscattered blood islands in Dear^(−/−) embryos but markedlyunderdeveloped dorsal aorta and peripheral vasculature, suggestingdeficiencies in primary vascularization despite the presence of bloodislands (FIG. 8A-F). Analysis of cardiac development in adjacentlittermates reveals that Dear^(−/−) hearts have poor cardiac chamberformation and endocardial cushion formation (FIG. 8A-F), consistent withincomplete cardiac looping observed on analysis of whole embryos (FIG.6).

The Dear^(−/−) mutant vascular and cardiac phenotypes in the C57BL/6Jgenetic background (BC10) is similar to that observed in VEGF^(+/−)deficient embryos (18, 19), but quite distinct from that observed inpreviously reported ET-1, ET_(A) and ET_(B) receptor null embryos(10-12, 24), as well as AngII, AT1a, AT1b, and AT2 receptor null mutants(14-17). The similarity to VEGF^(+/−) deficient mouse vascular phenotype(18, 19) suggests that both VEGF and Dear-mediated signaling arenecessary for angiogenesis and vascular network development, as well asmodulate blood island formation. The fact that both VEGF and Dear nullmutations are embryonic lethal, along with other similarvascular-phenotype null mutants such as transforming growth factor-β1(25), suggest that multiple pathways are involved in vascular networkformation—all necessary but none sufficient. The slightly later butoverlapping range of embryonic lethality indicates that Dear-mediatedpathways are downstream to VEGF-mediated pathways. The association ofvascular network deficiency and arrest of cardiac development in bothVEGF^(+/−) and Dear^(−/−) deficiency suggests that vascular-derivedsignaling plays a role in the progression of the complex development ofthe heart into a multi-chambered pump. The finding that Dear^(−/−)deficiency results in embryonic lethal vascular network abnormalities,while ET-1^(−/−) inactivation does not interfere with vascularnetworking (10) implies the existence of an alternative ET-1 source thatis produced independently of the well known Pre-pro-ET-1 pathway (26) oralternatively, the existence of an [ET-1]-like ligand that activatesDear and underlies Dear-mediated angiogenic roles.

Methods

Northern Blot Analysis

Total RNA was extracted with TRIzol (Invitrogen, Life TechnologiesInc.,) from the different tissues analyzed. PolyA⁺ RNA was subsequentlyisolated from total RNA using the Dynabeads mRNA purification kit (DynalBiotech) as per manufacturer's specifications. PolyA⁺ RNA (3 μg) was runon a 1% formaldehyde-denaturing agarose gel, transferred to Zeta-Probeblotting membrane (Bio-Rad) and UV cross-linked prior to hybridization.Hybridization was done in a buffer containing 5×SSC, 20 mM Na₂HPO₄, 7%SDS, 1×Denhardt's, 100 ug/ml denatured Calf thymus DNA at 50° C. for 24h. A γ-³²P-end labeled anti-sense mouse Dear oligonucleotide(5′-AGT-GAT-AGA-GCC-CCA-GTT-CCA-GCG-AGA-CTT-CAT-CTC-CTT-GC-3′ (SEQ. ID.NO. 8)) was used as probe. Membranes were washed sequentially with3×SSC, 5% SDS at 50° C. two times for 30 minutes each, and once with1×SSC, 1% SDS at 50° C. for 30 min. Autoradiography was carried out at−80° C. with intensifying screen.

Characterization of 129SVJ Mouse Dear Gene

One million independent recombinants from a λFIXII 129SVJ mouse genomiclibrary were screened with the full-length 3274 bp rat Dear cDNA¹ asprobe. Six independent genomic clones were identified andplaque-purified after a fourth round of screening. One of them, λ191,was characterized further by restriction digestion and subsequentsouthern blot analysis. A single 8 kb BamHI/BamHI restriction fragment(that hybridized to the 3274 bp rat Dear cDNA probe) was subcloned intopsp73 plasmid vector and sequenced.

Targeted Disruption of Dear in Mice and Production of Chimeric Mice

All animal procedures were performed in accordance with institutionalguidelines. A targeting vector was constructed by replacing a 300-bppiece containing the 3′-end of Dear with the PGKNeo-cassette (FIG. 5)effectively deleting amino acids 81-127 of Dear. The targeting vectorwas electroporated into 129SVJ ES cells and G418 resistant cell cloneswere isolated. Genomic DNA was obtained from each ES cell clone,restricted with SphI and subjected to Southern blot analysis. A 1.5 Kbfragment of Dear was used as probe (FIG. 5). The presence of an 8 Kbendogenous band and a 5.2 Kb band was indicative of homologousrecombination (FIG. 5). Homologous recombination was further verified byPCR analysis using an upstream primer (P1: 5′-TGTGAGGCTAGAAGGCTGC-3′(SEQ. ID. NO. 9)) located 171 bp upstream from the 5′-end of thetargeting vector and a reverse primer (P2: 5′-GAGCAAGGTGAGATGACAGG-3′(SEQ. ID. NO. 10)) located in the PGKNeo cassette (FIG. 5).Amplification of a 5.5 Kb fragment that hybridized to the same probeused in the Southern blot analysis (FIG. 5) was indicative of homologousrecombination. Five positive ES cell clones were then microinjected into129SVJ blastocysts generating 14 chimeric mice that were used toestablish the Dear knockout line. Speed-congenic backcross breeding toinbreed onto C57BL/6 genetic background was done for more than tengenerations, ≧BC10, providing all Dear^(+/−) and Dear^(−/−) mice foranalyses (>99.95% congenic line in C57BL/6 background).

Characterization of Mouse Dear cDNA and Expression Studies

Mouse Dear cDNA was obtained by RT-PCR from C57BL/6 mouse kidney PolyA⁺RNA (forward primer, 5′-CACACAAAGCCTTACTTTATCC-3′ (SEQ. ID. NO. 13);reverse primer, 5′-AAAGCCAGCCTTTAGATAACC-3′(SEQ. ID. NO. 14)), subclonedinto the PT-vector system (Clontech, Palo Alto, Calif.) and thensequenced (GenBank accession no. DQ009865). RNA blot analysis was doneas described (1) using PolyA⁺ RNA (3 μg), γ-³²P-end labeled anti-sensemouse Dear oligonucleotide(5′-AGTGATAGAGCCCCAGTTCCAGCGAGACTTCATCTCCTTGC-3′(SEQ. ID. NO. 15)) asprobe. Receptor expression studies, ¹²⁵I-ET-1-1 and ¹²⁵I-AngII bindingto membranes were done as described (2).

Genotyping of Mouse Embryos

Genotyping was done by PCR analysis of genomic DNA isolated fromextraembryonic membranes. Primers flanking the Sad site localized withinthe amino acid coding region of Dear (upstream primer:5′-AACTTCTCTGGTCCGCTCC-3′ (SEQ. ID. NO. 11); downstream primer:5′-ACTTGCTGAAACTAAAACCTGC-3′ (SEQ. ID. NO. 12)) were used to detect thewild type allele (PCR product=153 bp indicative of the presence of thewild type allele) and primers P1 and P2 (described above) to detect themutated allele (PCR product=5.5 Kb indicative of the presence of themutated allele).

Analysis of Heterozygous Dear^(+/−) Phenotype

We analyzed backcross BC10[C57BL/6] Dear^(+/−) mice for Dear proteinlevels by Western blot analysis using equal amounts of protein (40 μg)from mouse kidney membranes and rabbit IgG anti-mouse Dear anti-peptidespecific antibody (1:500 dilution) developed against mouse Dear specificsynthetic peptide: L₁₆SKCNHNEQDTA₂₇ (SEQ. ID. NO. 16) to detectDear-specific polypeptide. We measured blood pressure in 6 month oldmice by tail-cuff sphygmomanometer (Visitech BP 2000, Visitech CA) underlight anesthesia ascertaining equivalent physiologic state by limitingBP measurements to periods with heart rate ranging from 300-500 beatsper minute. We obtained three sets of 10 consecutive readings per mouseand took the average of at least 20 readings within the prescribednormal heart rate range.

Histology

Embryos were collected at embryonic E9.5-E12.5 days from timed-pregnantmice (counting noon of the day a vaginal plug is detected as E0.5);genotypes were determined by PCR analysis of extraembryonic membranetissue DNA. Embryos were analyzed and photographed within their yolksacs, then fixed in 4% freshly prepared PBS-buffered paraformaldehyde.Histology processing and Masson-trichrome staining were done followingestablished procedures. Digital stereophotomicroscopy and bright-fieldphotomicroscopy were done using a Nikon stereomicroscope and ZeisAxioskop microscope respectively. Immunohistochemistry was doneessentially as described (27).

Example 3 Blood Pressure Measurements

Male and female cohorts of Dear KO and wild type (N10 backcrossgeneration) were used to measured BP. Twelve (+/−) and 11 (+/+) femalemice and 14 (+/−) and 14 (+/+) male mice were studied. Testing was doneat 6 months of age.

Mice were maintained on regular rodent chow and on a 12-hour light/darkcycle. Mice were transported and allowed to settle in the procedure room1 hour before measurements were taken. Systolic BP along with heart ratewas measured by a programmable tail-cuff sphygmomanometer (Visitech BP2000, Visitech, NC). Mice were lightly anesthetized with intraperitonealketamine (80 mg/kg) and xylazine (18 mg/kg) and placed on the heatedplatform after a 2 minute interval. Three sets of 10 consecutivereadings each were taken per mouse. Data is presented as average of atleast 20 readings per mouse spanning the heart rate range of 300-500bpm.

Results

As shown in FIG. 10A, SBP did not differ between WT and KO male mice(WT, 137.2±5.1; KO, 138.6±3.7; t=0.05, P>0.8) at 6 months of age. Incontrast, SBP is significantly lower in KO female mice when comparedwith WT female mice (WT, 134.9±4.0; KO, 112.3±6.1; t=3.03, P<0.01). Meanheart rates did not differ between contrasting groups (FIG. 10B)affirming the SBP differences observed between WT and KO female mice.Thus, heterozygosis at the Dear locus shows gender-specific effects onBP affecting only females. This result is consistent with recent datasuggesting a female-specific effect of Dear variants in salt-sensitivehypertension in the Dahl rat model (2).

Example 4

We next investigated the role of Dear-inhibition in two establishedrodent tumor models. First, comparing heterozygous Dear^(+/−) deficientmice and wild type Dear^(+/+) littermates, we detect significantreduction in tumor mass (FIG. 11A-B) and tumor volume (FIG. 11C-D) inB16-F10 melanoma cell-induced tumor model (74) in heterozygousDear^(+/−) deficient-female (t-test, P<0.02) mice but not in male mice.Secondly, because effects were seen only in female Dear^(+/−) mice, wenext tested whether Dear-inhibition would reduce tumor growth in¹³⁷Cs-radiation induced breast cancer model (70) in female rats withtumor latency less than 3 months. Using two independent inhibitionmethods, anti-rat Dear anti-peptide specific antibody begun 4 weeksafter irradiation (FIG. 11C) and anti-rat Dear DNA vaccine begun twoweeks after irradiation (FIG. 11D), we detect significant reductions intumor growth during a 6-week observation period. In contrast torespective control groups, both anti-Dear treatments prevented tumorgrowth with significant reductions in %-change in tumor volume detectedfrom 4-6 weeks after tumor appearance (ab-Rx: P<0.05-0.01; DNA-v:P<0.02-0.001). Furthermore, we detect significant tumor regression with68% reduction in tumor volume in anti-Dear DNA-vaccinated rats (FIG.11D, P<0.01). Inhibition of tumor growth is associated with decrease inmalignancy-potential based on tumor pattern, nuclear grade and vascularinvasion in both anti-Dear antibody and DNA vaccine treated ratscompared with age-matched, non-treated control rats (FIG. 11E).Furthermore, decrease in the number of chaotic and mosaic vessels wasalso detected as a result of anti-Dear treatment.

Methods

Tumor Studies and Dear-Specific Inhibition

We developed B16-F10 (ATCC) melanoma cell-induced subcutaneous tumormodel essentially as described (74) in 10-week old Dear^(+/−) andlittermate Dear^(+/+) male and female mice (n=5 per group). Thirteendays after tumor induction, we excised tumors and measured tumor weightand volume. We induced rat mammary gland tumors in 48 Sprague Dawleyrats (n=12 per group) essentially as described (70) at 40 days of agevia ¹²⁷Cs-radiation. Only rats with tumor latency less than 3 monthswere used for study (n=4 for anti-Dear anti-peptide antibody; n=3 forcontrol antibody; n=3 for anti-Dear DNA-vaccine; n=3 for control pcDNA).We began antibody treatments 6 weeks after irradiation at 12 weeks ofage. We used affinity-purified rabbit IgG anti-Dear anti-peptideantibody raised against the synthetic peptide P₅₁LLTSLGSKE₆₀ (1) (SEQ.ID. NO. 7). As control, we used rabbit IgG (sc-2027, Santa CruzBiologicals, Santa Cruz Calif.). Test and control antibodies wereinjected thrice weekly intraperitoneally (6 μg/injection) until the endof the study at 6 weeks post tumor appearance. In parallel, we injectedtest and mock-DNA vaccines—using anti-Dear (pcDNA-Dear) DNA-vaccine andcontrol expression vector (pcDNA, Invitrogen, Carlsbad, Calif.)mock-vaccine—two weeks after irradiation at 8 weeks of age, andthereafter bi-weekly until 6 weeks from tumor appearance (500 μg perdose, intramuscularly). Tumor volume was measured using the formula(4/3πr₁ ²×r₂) where r₁ is the smaller, and r₂ the larger radius asdescribed (71). We used t-test, two-way repeated measures ANOVA andTukey's post test for multiple pairwise comparisons for statisticalanalysis as appropriate.

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All references described herein are incorporated herein by reference intheir entirety.

TABLE 1 Ligand affinities for Dear S44P/M74T and S44/M74 variantsB_(max), Variant pmol/mg K_(H), nM K_(L), nM h ¹²⁵I-[Tyr⁴]Angiotensin IIS44P/ no binding M74T S44/M74  23.6 ± 0.92 0.23 ± 0.08 2.65 ± 0.11 2.63± 0.25 ¹²⁵I-[Tyr¹³]Endothelin-1 S44P/ 20.21 ± 1.59 12.0 ± 1.12  836 ±38.1 1.45 ± 0.09 M74T S44/M74 26.25 ± 1.29  4.42 ± 0.89*   450 ± 12.4*1.41 ± 0.12 Values are means ± SE, B_(max), maximum ligand binding sitesin pmol/mg membrane protein; K_(H), dissociation constant for highaffinity binding site; K_(L), dissociation constant for low affinitybinding site; h, Hill coefficient. *P < 0.01 (t-test).

TABLE 2 Chromosome 2 analysis of F2(R × S) male and female cohorts F2(R× S) Males Females LRS TTV, % P LRS TTV, % P MAP Locus Marker, cM(males/females) 134.3 (1 3.1) 122.6 (10.4) D2Rat124 0.0/0.0 1.8 20.40800 1.2 1 0.55024 D2Rat196 9.1/7.2 0.7 1 0.71099 1.1 1 0.57694D2Rat19 14.1/13.5 0.9 1 0.62681 2.5 2 0.29225 D2Rat21 8.4/6.1 1.3 10.52159 3.4 3 0.17980 D2Rat143 12.3/14.9 1.6 1 0.45032 11.4 11 0.00332D2Rat161 25.2/10.8 0.2 0 0.91259 8.8 8 0.01247 D2Rat34 25.8/12.4 4.3 40.11848 11.2 10 0.00376 Dear 14.8/14.6 6.1 6 0.04816 15.2 14 0.00050D2Mgh11/ 2.9/6.1 7.7 7 0.02155 9.6 9 0.00840 ATP1A1 D2Rat169 7.8/4.5 6.26 0.04520 5.4 5 0.06632 D2Rat59 10.1/8.8  2.7 3 0.26394 1.8 2 0.41202SBP 157.2 (14.2) 145.0 (11.4)

TABLE 3 Table 3. Analysis of S allele effects on blood pressure of Dearlocus in F2(R × S) intercross rats BP, mmHg (±sd) Tukey Test P SS SR RRANOVA P SS vs. RR SS vs. SR SR vs. RR S Allele Effect Females MAP 130(13.1) 122 (9.1) 119 (8.3) 6.2 × 10⁻⁴ 4.8 × 10⁻⁴ 0.01 NS ? SBP 152(14.2) 145 (10.0) 141 (9.7) 1.7 × 10⁻³ 1.0 × 10⁻³ 0.02 NS ? DBP 109(12.0) 102 (8.7)  99 (7.6) 7.3 × 10⁴ 5.5 × 10⁴ 0.01 NS ? Males MAP 139(13.6) 133 (13.4) 131 (10.0) 0.052 NA NA NA SBP 162 (14.4) 156 (14.7)152 (10.9) 0.034 0.04 NS NS ? DBP 117 (12.6) 113 (12.0) 110 (9.2) 0.085NA NA NA Values are means, with SD in parentheses. ANOVA, analysis ofvariance; Tukey test, all pairwise multiple comparison procedure; NS,not significant; NA, not applicable.

1. A method for determining an individual's susceptibility tohypertension comprising: (a) obtaining a biological sample from apatient; and (b) detecting the presence or absence of at least onemutation or polymorphism in the Dual Endothelin-1/Angiotensin IIReceptor (Dear) in said tissue sample as compared to a control sample,(c) determining whether the mutation or polymorphism increases theexpression of Dual Endothelin-1/Angiotensin II Receptor (Dear), enhancesthe affinity of ET-1 binding to Dual Endothelin-1/Angiotensin IIReceptor (Dear), enhances the affinity of VEGF-signal peptide (VEGFsp)binding to Dual Endothelin-1/Angiotensin II Receptor (Dear), orincreases Dear-activation when Dear is stimulated with a ligand, whereinthe presence of at least one of the mutations or polymorphisms indicatesthat the individual is susceptible to hypertension.
 2. A method fordiagnosing hypertension comprising: (a) obtaining a biological samplefrom a patient; and (b) detecting the presence or absence of at leastone mutation or polymorphism in the Dual Endothelin-1/Angiotensin IIReceptor (Dear) in said tissue sample as compared to a control sample;(c) determining whether the mutation or polymorphism increases theexpression of Dual Endothelin-1/Angiotensin II Receptor (Dear), enhancesthe affinity of ET-1 binding to Dual Endothelin-1/Angiotensin IIReceptor (Dear), enhances the affinity of VEGF-signal peptide (VEGFsp)binding to Dual Endothelin-1/Angiotensin II Receptor (Dear), orincreases Dear-activation when Dear is stimulated with a ligand, whereinthe presence of at least one of the mutations or polymorphisms indicatesthat the individual has hypertension.
 3. The method of claim 1 furthercomprising performing a polymerase chain reaction (PCR) to amplify theDEAR coding sequence.
 4. The method of claim 2, wherein the DEAR codingsequence is a human DEAR coding sequence and has at least 75% homologyor identity to SEQ ID NO.
 1. 5. The method of claim 1 furthercomprising: (a) isolating nucleic acid from the biological sample; and(b) contacting the nucleic acid with at least one nucleic acid probeunder selective hybridization conditions, wherein said probepreferentially hybridizes with a nucleic acid sequence comprising a Dearmutation or polymorphism, wherein the binding of the probe to theisolated nucleic acid indicates that the individual is susceptible to orcurrently has hypertension.
 6. A method for enhancing angiogenesis at aclinically relevant site in an individual comprising administering tosaid patient an effective amount of a Dual Endothelin-1/Angiotensin IIReceptor (Dear) activator.
 7. The method of claim 7, wherein saidclinically relevant site is selected from the group consisting of awound, ulcer, diabetic ulcer, and a heart with coronary artery disease.